SUGGESTED ANSWER FOR TUTORIAL 5

QUESTION 1

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.

a) Describe the order of elution for the above separation.

(5 marks)

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

Followed by neutral species which they eluted together as they will not separated;

Anions eluted last, even though 20Y- has greater charge to mass ratio and greater
the electrophoretic force but towards opposite direction. Thus it eluted later than
25 -
Y.
20
Order of elution: X+, 25X+, A & B, 25Y-, 20Y-

b) Explain what would happen to the separation if:

i) the pH was decreased to 4.0

(2 marks)

At pH 4.0, the silanols on the capillary wall were less ionized compared to
at pH 5.8, therefore the EOF was reduced, and this decreased the migration
rate of analytes but the elution order was remained.

ii) the pH was decreased to 2.0 (assume that the charges of the components do not
change).
(3 marks)

When the pH below 3, the capillary wall was not ionized. Therefore
migration based on electrophoretic flow, whereby the ions migrated toward
electrode with opposite charge and the neutrals were not attracted to either
electrode. Since detector was at the cathode (-ve), only cations were
separated and detected based on charge to mass ratio.

iii) the pH was 5.8 and treated with ethyl ammonium bromide.
(3 marks)

The ammonium cation become attached to the negatively charged silica

surface and, in turn, create a negatively charged doubled layer of solution,
which is attracted toward the anode, thus reversing the EOF. The elution
order reversed but all the analytes were not detected as the detector was
not anode.
 iv) the pH was 5.8 and added with high concentration of anionic surfactant (above
critical micellar concentration/CMC level).
(3 marks)

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.

(Q5 OCT 2010)

QUESTION 2

a) What is electroosmotic flow? Explain the effect of pH on elecroosmotic flow.

(4 marks)

EOF is a net flow (or bulk flow) of buffer solution in the capillary towards one
single direction under an electric field.

EOF mobility is 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
increased, the magnitude of EOF is increased.

b) Outline how a capillary electrophoresis can be modified to allow efficient separation of:

i) neutral molecules

Surfactant (example SDS) is added to the buffer solution above its critical
micelle concentration (CMS), so micelles are formed. Micelles are formed
with the hydrophobic (hydrocarbon) tails pointing inward and the
hydrophilic (negatively charged) heads pointing outward into the aqueous
solution. The interior of the micelles is non-polar, therefore capable to
absorb non-polar (neutral) analytes into the hydrocarbon tails of the
particles and solubilizing the analytes.

ii) anions

Treat the capillary with cationic surfactant (example Ethyl ammonium

bromide) to reversed the EOF towards anode and position the detector at
anode and injection at cathode.

(8 marks)

c) Discuss a method that is best for the separation of macromolecules such as proteins
and DNA fragments that have similar charges but differ in size.
(4 marks)

Capillary gel electrophoresis (CGE). The separation in CGE is carried out in a

porous gel polymer matrix, the pores of which contain the buffer mixture. These
provide a molecular sieving action that retarded the migration of analyte species
 to various extents depending upon the pore size of the polymer and the size of the
analyte ions.
(Q5 JAN 2012)
QUESTION 3

a) The separation of a mixture of a cation, a neutral species and an anion was conducted
using capillary electrophoresis.

i) Discuss the experimental conditions required so that separation can be achieved

and state the order of elution.
(4 marks)

- pH must be above 3 for EOF. EOF will drag all the species to the cathode.
- detector must be at the cathode so that all can be detected.

ii) State the order of elution of these analytes.

(2 marks)
Elution: cation < neutral < anion

b) For each of the following capillary zone electrophoresis separation, explain the reason
for the mentioned problems and suggest an approach to improve the separation.

i) In the separation of two neutral species (A: non polar, B: intermediate polarity)
and an anion (50Y-) conducted at pH 5, only two peaks were observed.
(4 marks)

One peak for anion and two neutrals are not separated and eluted as one
peak since both do not have any charge (1).

Add surfactant to form micelles (1) and able to absorb nonpolar

compounds into the hydrocarbon interior of the particles (1), thus
solubilizing the nonpolar compound and the two neutrals can be separated.

ii) In the separation of 50X+ and 55X+ conducted at pH 9, the migration time of the
two compounds were too close, producing overlapping peaks.
(4 marks)

For cations, the EOF is the same direction as the movement of ions (1),
therefore at high pH the EOF is very strong and causes small difference in
migration time of the two compounds (1). Reduce pH but must be above 3
to reduce the EOF and causes the two compounds to be separated (2).

(Q4 JUNE 2012)

QUESTION 4

a) Capillary electrophoresis (CE) is a powerful separation technique. In CE, analytes

migrate in a buffer solution through a capillary based on two principles: electrophoretic
flow and electroosmotic flow (EOF).

i) Explain the differences between electrophoretic flow and EOF.

(4 marks)
 Electrophoretic flow caused by the presence of an electric field that has
been applied to the buffer solution across the capillary column (1).

Charged molecules migrate in the direction of the electrode bearing the

opposite charge (1).

EOF is a net flow (or bulk flow) of buffer solution in the capillary towards
one single direction under an electric field (1).
Above pH3, the capillary wall will be ionized and formed electric double
layer of net buffer solution (cation, neutral, anion) toward cathode (1).

Ii) EOF is pH dependent. Explain why and how this will affect the migration rate of
cations.
(3 marks)

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 (1).
If the pH is increases, the magnitude of EOF increased (1) and thus
increase the migration rate (movement of cations are same direction with
EOF) (1)

iii) Explain how EOF can be manipulated to separate anions efficiently.

(4 marks)

The EOF is reversed by treating the walls of the capillary with a cationic
surfactant (alkyl ammonium salt such as ethyl ammonium bromide)(1).
The surfactant absorbs on the capillary wall and makes the wall positively
charged (1).
Buffer anions congregate near the wall and are swept towards the anode,
thus reversing the EOF.
The detector must be placed at anode end and the sample introduction at
cathode end (1).

b) A separation of neutral compounds in micellar electrokinetic capillary chromatography

(MEKC) is based on varying hydrophobicities. Explain.
(4 marks)

Surfactants are added to the buffer solution at concentrations that form micelles.

Above a certain concentration, called the critical micelle concentration (CMC),

the surfactant molecules will self-aggregate, forming micelles in aqueous solution
(1).

The interior of the micelles is non-polar, therefore capable to absorb non-polar

(neutral) analytes into the hydrocarbon tails of the particles and solubilizing the
non-polar analytes (1).
 The more time the neutral molecule spends inside the micelle (retain), the longer
is its migration time (1).

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 (1).

c) Capillary gel electrophoresis (CGE) is commonly used to analyze DNA fragments.

Explain.
(4 marks)

CGE provides a molecular sieving action (1) that retarded the migration of
analytes to various extent depending upon the pore size of the polymer in column
and the size of the analytes ions (1).
The sieving action is particularly helpful in separating macromolecules such as
protein and DNA fragments that have substantially the same charge but differ in
size (2).

(Q4 JAN 2013)

QUESTION 5
 (Q4 JUNE 2013)
a) i) Rate of migration = electroosmotic + electrophoretic (1).
Above pH 3, EOF towards negative electrode (1).
For electrophoretic, all the cations move towards negative electrode (1).
Arsenic compounds with higher charge/mass eluted earlier (1).

ii) The positively charged ammonium ions become attached to the negatively
charged silica surface and in turn, create a negatively charged doubled
layer of solution (1), which is attracted toward the anode (1), thus reversing
the EOF (1).

For electrophoretic, all are cations move towards negative electrode (1).
Therefore, arsenic compounds with lower charge/mass eluted earlier (1).

ii) Addition of surfactant to form micelles (1). Micelles form in aqueous

solution when the concentration of an ionic species having a long-chain
hydrocarbon tail is increased above a certain level called the critical
micelle concentration (CMC) (1) whose hydrocarbon tails are in the interior
of the aggregate (1) and whose charged ends are exposed to water on the
outside (1). Micelles are capable to absorb neutral compounds into the
 hydrocarbon interior of the particles depending on the polarity of the
compounds and move with the micelles (1).

Hydrophilic neutral will be at the exterior of the micelles and move towards
negative electrode first (1). Hydrophobic neutral will enter the micelles and
eluted later (1).

b) This sieving action (1) with smaller compounds eluted earlier than bigger
compounds (1). Thus able to separate macromolecules (large molecular weights)
(1) that have substantially the same charge but differ in size (1)
(Q4 JUNE 2013)

QUESTION 6

The separation of a mixture of 2 cations (X+ and Y2+) and 2 anions (M- and N2-) was done using
capillary electrophoresis (CE). All species are of comparable masses.

a) Electrosmotic flow (EOF) is a unique feature in electrophoresis. Explain the existence of

EOF.
(3 marks)

Above pH3, the capillary wall will be ionized (1) and formed electric double layer of
net buffer solution (1) (cation, neutral, anion) toward cathode (1).

b) In the above electrophoresis analysis, a capillary column treated with cetyl

tyrimethylammonium bromide was used and the analysis was done at pH 6. Draw a
labeled schematic diagram of the CE instrumentation showing the placement of the
injector, detector and the direction of the EOF.
(5 marks)
Treated: Reversed EOF (1)
Direction of EOF flow is cathode to anode (1)
Injector at cathode end (1)
Detector at anode end (1)
Correct diagram and label (1)
c) State the order of elution for the components in (b). Briefly explain the reason for the
chosen order.
(4 marks)
N2-, M- , X+, Y2+ (2)
The migration rate of the ions is dependent on the charge to mass ratio. Anion
N2-, higher ration than M-, therefore reach detector first (1).

Since the EOF is towards the anode the electrophoretic flow of cations has
negative value hence X+ (lower ratio) will elute faster than Y2+ (higher ratio) (1).

d) Using the above CE method, separation of a mixture of two neutral species, P

(hydrophobic) and Q (hydrophilic), could not be achieved. Explain why. Discuss another
CE approach to separate these neutral species
(4 marks)

The 2 neutrals will migrate at the same rate with the EOF, thus no separation (1).
 Use MEKC (1). With the addition of micelles (1), the hydrophobic species will be
absorbed into the micelles and move together with the micelle. The hydrophilic
neutral will stay in the buffer (1).
(Q4 DEC 2013)

QUESTION 7

a) In capillary electrophoresis (CE), electroosmotic flow (EOF) determines the migration

rate of ionic species. For the analysis of anions, discuss the factor that can:

i) Increase the magnitude of EOF.

pH (1). If the pH is increases (pH >3) , charge density increases, the

magnitude of EOF increased (2).

ii) Reverse the direction of EOF.

The EOF is reversed by treating the walls of the capillary with a cationic
surfactant (alky ammonium salt such as ethyl ammonium bromide)(1).
The surfactant absorbs on the capillary wall and makes the wall
positively charged (1). Buffer, anions congregate near the wall and are
swept towards the anode, thus reversing the EOF (2).

b) When capillary zone electrophoresis (CZE) was applied in the separation of three
cations and two hydrophobic neutral species, only four peaks were observed. Explain.
Suggest an alternative electrophoresis approach to separate all the species.
(5 marks)

Hydrophobic neutral species are separated as one peak only (1). Only MEKC can
separate neutral and ionic species (1). With the addition of micelles (1), the
hydrophobic species will be absorbed into the micelles and move together with
the micelle (1). Partitioning of the neutral analytes based on their varying
hydrophobicities (1). More hydrophobic spend more time inside the micelle,
migrate slowly cause longer migration time compared to less hydrophobic (1).

(Q4 JUNE 2014)