AS/OCT2009/CHM510

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UNIVERSITI TEKNOLOGI MARA

FINAL EXAMINATION

COURSE

ANALYTICAL SEPARATION METHODS

COURSE CODE

CHM510

EXAMINATION

OCTOBER 2009

TIME

3 HOURS

INSTRUCTIONS TO CANDIDATES
1.

This question paper consists of six (6) questions.

2.

Answer ALL questions in the Answer Booklet. Start each answer on a new page.

3.

Do not bring any material into the examination room unless permission is given by the
invigilator.

4.

Please check to make sure that this examination pack consists of:
i)
ii)

the Question Paper

an Answer Booklet - provided by the Faculty

DO NOT TURN THIS PAGE UNTIL YOU ARE TOLD TO DO SO

This examination paper consists of 4 printed pages
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AS/OCT 2009/CHM510

QUESTION 1
a)

Why is eddy diffusion not a concern when using capillary columns?

(2 marks)

b)

Discuss the effect of the particle size on the separation performance in packed
column liquid chromatography.
(6 marks)

c)

For a separation of compound A and B on a chromatographic column, the retention

time for peak A and peak B were 2.0 min and 2.5 min respectively. The dead time
was 0.5 min.
i)

Calculate the capacity factors for peak A and peak B.

(4 marks)

ii) Calculate the peak width of A if the number of theoretical plate height was
40,000.
(3 marks)

QUESTION 2
Explain how temperature programming (in gas chromatography), gradient elution (in liquid
chromatography, and pressure programming (in supercritical fluid chromatography) can be
used to optimize a separation for maximum efficiency and minimum analysis time. Use
appropriate example in your discussion.
(15 marks)

QUESTION 3
For each of the following analysis, discuss the reason for the underlined parameter/
approach used.
a)

In the analysis of polyaromatic hydrocarbons from contaminated water, gas

chromatographic separation was done using polvdimethvl siloxane column and
splitless injection mode.
(5 marks)

b)

The unknown alkaloids isolated from plants were analysed using gas
chromatography with mass spectrometry detector. The extract was derivatized prior
to gas chromatographic analysis.
(5 marks)

c)

Carbon dioxide is the mobile phase most commonly used in supercritical fluid
chromatography (SFC). However, for the analysis of relatively polar compounds,
methanol is added in a small concentration into the mobile phase system.
(5 marks)

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d)

AS/OCT 2009/CHM510

For the analysis of thermally labile volatile compounds, separation can be conducted
using gas chromatograph with on column injection. However for the analysis of
thermally labile non volatile compounds a supercritical fluid chromatography is the
best choice.
(5 marks)

QUESTION 4
For each of the following, discuss the reason for the mentioned problem and suggest an
approach to overcome the problem.
a)

In a separation using a C18 column with a mobile phase of acetonitrile/water 80/20,

the first two peaks eluted very close to tM.
(4 marks)

b)

7-chlorophenol and 2-chlorophenol were not separated on a bonded siloxane with

diol functional groups stationary phase.
(4 marks)

c)

In the analysis of cations in water sample using single column ion chromatography,
most of the cations could not be detected due to the low sensitivity of the conductivity
detector
(4 marks)

d)

The cations in a water sample at pH 2 cannot be separated on a carboxylic acid

resin.
(4 marks)

e)

When a Bio-gel 150 with a molecular mass range of 15,000 - 150,000 is used for
size exclusion chromatography packing, protein A (MW 185,000) and protein B (MW
158,000) are not retained and eluted together as a single peak.
(4 marks)

QUESTION 5
a)

Ions in a capillary move as a result of electrophoretic flow and electroosmotic flow

(EOF). Suggest two different approaches (other than reducing the voltage) to reduce
the electroosmotic flow.
(4 marks)

b)

The water soluble vitamins; niacinamide (a neutral compound), riboflavin (a neutral

compound), and thiamine (a cation) were separated by micellar electrokinetic
capillary chromatography (MEKC) in 15 mM borate buffer (pH 8) with 50 mM sodium
dodecyl sulfate. The migration times were niacinamide (8.1 min), riboflavin (13.0
min) and thiamine (14.9 min)

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c)

AS/OCT 2009/CHM510

i)

Explain why niacinamide and riboflavin (both are neutral compounds) have
different migration times.
(3 marks)

ii)

Explain what will happen to the migration of the above vitamins if sodium
dodecyl sulfate was added in a very low concentration (< 50 mM) into the
system.
(3 marks)

Describe the principle of separation in capillary gel electrophoresis.

(4 marks)

QUESTION 6
a)

Solid phase microextraction (SPME) utilized a fused silica fibre usually coated with a
polymeric phases. The analytes from the sample are adsorbed by the fibre until
equilibrium is reached in the system. The analytes are then desorbed from the fibre
prior to analysis.
i)

How to determine the amount of analytes adsorbed by the fibre coating at

equilibrium?
(2 marks)

ii) Describe two approaches for desorption.

(4 marks)
iii) Give two parameters that may affect the efficiency of the extraction in SPME.
(2 marks)
b)

Both pressurized liquid extraction (PLE) and supercritical fluid extraction (SFE) are
techniques developed for the extraction of solid and semi solid samples.
i)

Compare both techniques in terms of volume of organic solvent and quality of

extracts.
(2 marks)

ii) Discuss how the properties of supercritical fluid affect the efficiency of SFE.
(3 marks)
iii) Discuss how the temperature affects the efficiency of PLE.
(3 marks)

END OF QUESTION PAPER

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