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DATE: 6 October 2017 DURATION: 1HR



1.1 “Electrophoresis is a fundamentally different separation technique than chromatography”.

Briefly describe the fundamental difference between the 2 separation techniques?

Chromatographic separations are based on partition differences of the analytes between

the stationary and mobile phases.
Electrophoresis is based on differences in migration of charged particles in an electric


1.2 List the 4 basic components of a CE instrument

consists of an injection system,

a separation capillary (20–100 mm I.D., 20–100 cm length),
a high voltage source (delivering up to 30 kV and up to 200–250 mA),
electrodes and electrode jars and detector(s).

1.3 The following bullet points describe the principles of separation in CE. You are required
to fill in ONLY the missing word labeled (A-E).

 Electrophoresis takes place in a buffer filled, narrow-bore____________ A capillaries.

• Each________ A capillary is about 25-100 μm in internal diameter
• When a_______ B voltage is applied to the solution, the molecules _________ C move
through the solution towards the electrode of __________ D opposite charge
• Depending on the charge, the molecules___________ C move through________ A at
_______E different speeds.

1.4 Explain what is meant by electro-osmotic flow (EOF) (3)
EOF is the bulkflow of liquid in the capillary and is a consequence of the surface charge on the interior
capillary wall. The fused silica capillaries used for separations have ionizable silanol groups in contact
with the buffer contained within the capillary.
The EOFresults from the effect of the applied electric field on the solution doublelayer at the wall. The EOF
controls the amount of time solutes remain in the capillary by superposition of flow on to solute mobility.
This can have the effect of altering the required capillary length, but does notaffect selectivity.

1.5 A vitamin sample with a mixture of 3 analytes comprising of a positive, negative and a
neutral charge was separated with a positive polarity. The effective and total lengths of
the capillary are 50 and 58.5 cm respectively, with a diffusion coefficient of 1 x 10-6 cm2/s
and an applied voltage of 25 kV. The tabulated data below was extracted from the

Solute Migration Time/min

Cationic 1.717
Neutral 1.895
Anionic 13.238

1.5.1 Use vector diagrams to describe the order of elution. (3)

1.5.2 Calculate the apparent mobility of the cationic species.

lL 50 x58.5
a    1.1x103 (2)
Vta 25000 x1.717 x60

1.5.3 Calculate the electro-osmotic flow (EOF) mobility of the neutral species and comment on
the significance of your answer.

lL 50 x58.5
eof    1.0 x103
Vt N 25000 x1.895 x60
The neutral species are carried by the same velocity of the EOF. The EOF controls
the amount of time solutes remain in the capillary by superposition of flow on to the
solute mobility.

1.5.4 Calculate the number of theoretical plates.

V 1.1x103 x 25000
N   1.51x107
2D 2 x1.0 x106


1.6 order is from top to bottom, due to increasing size of the molecules. (2)