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QUESTION 1
ii) Why is silica stationary phase generally limited to operating in the pH range of
2-8?
(2 marks)
iv) Explain briefly how the retention times would be affected if 80% methanol were
used instead.
(2 marks)
ii) Peaks continue to elute for a considerable time after completion of the gradient.
(3 marks)
(4 marks)
Total permeation limit defined as the limit which molecules that are smaller
than the pore size can enter all pores (1) and elute together as the last peak
in the chromatogram (1) ;
Molecules larger than the pore size cannot enter the pores (1) and elute
together as the first peak in the chromatogram (1) is called total exclusion
limit.
QUESTION 2
i) the composition of the mobile phase is changed to 70% methanol and 30%
water
(3 marks)
ii) the C18 bonded phase contained unreacted silanol groups. Name the process
to reduce the concentration of unreacted silanols.
(3 marks)
b) Draw and label completely a chromatogram for the following sugar compounds
separated on a '500 A' column with a fractionation range of 50 - 1000: xylose
(MW150), glucose (MW 180), maltose (MW 342), maltoheptaose (MW 1153) and
starch (MW >1500). What are the exclusion limit and the permeation limit for this
separation?
(5 marks)
QUESTION 3
For each of the following, discuss the reason for the mentioned problem and suggest an
approach to overcome the problem.
Adsorption chromatography
Mobile phase : A non polar solvent (e.g. hexane) modified with a small
amount of polar solvent (e.g. acetone, diethyl ether, methylene chloride).
Note:
Mechanisme of the separation :
Analytes compete with mobile phase for the binding sites on the
stationary phase.
Analytes in the liquid sample interacts with adsorption sites on solid
surface.
Polar groups (-OH) on solid surface form dipolar interaction (e.g. H
bonds) with sample.
Analytes are separated based on repeated adsorption-desorption steps
onto the solid support.
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)
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 4
For each of the following, give a reason for the mentioned problem and suggest an
approach to overcome the problem.
(5 marks)
b) For the separation of organic acids using ion exchange chromatography, the
compounds were separated using carboxylic acid COO-H+ resin as the stationary
phase at pH 4. The compounds were not separated due to overlapping of peaks.
(5 marks)
c) You are given the task to separate anions in a water sample. In your laboratory,
the liquid chromatograph available can only run partition chromatography. When
the separation of anions in the sample was carried out using reverse phase
chromatography with methanol:water (50:50) as the mobile phase, no peak was
observed.
(5 marks)
Ion-pair chromatography (IPC)
Mechanism of separation :
The counter ion combines with the analyte ion to form an ion-pair(neutral
species/uncharged) that is retained by the RP packing.
Note:
IPC is a method for improving the separation of charged/ionized (cation &
anion) & very polar analytes on a reversed phase column.
The use of IP reagents can enhance peak shape & tR when common
remedies such as modifying eluent ratios or changing stationary phase fail.
d) When a Bio-gel 150 with a molecular mass range of 22,000 - 250,000 was used
for size exclusion chromatography packing, the first three compounds were
separated well. However, the last two compounds were eluted as a single peak.
(5 marks)