ANALYTICAL SEPARATION METHODS (CHM510)

Exercises
Q 1 (16 marks)
a) The chromatogram in Figure 1 shows the separation of 1mg/mL standards of chlorogenic acid,
caffeic acid and rutin using a 20 cm column. The peak widths for each compound are 0.72 min
(chlorogenic acid), 0.81 (caffeic acid) min and 1.36 min (rutin).

Figure 1

i) Calculate the linear velocity of rutin.

ii) Determine the number of plates of the column based on caffeic acid.

iii) mment on the separation of chlorogenic acid and caffeic acid by calculating resolution (Rs)

b) Table 1 tabulates the data obtained in the separation compound X from pharmaceutical product
using packed and capillary columns. Justify which column will give good separation.

Table 1

Compound Packed column Capillary column

Retention time Peak width (sec) Retention time Peak width
(sec) (sec) (sec)
Unretained 15 30
X 160 1.22 75 0.25

1
c) Figure 2 shows a van Deemter plot obtained using three columns of the same dimension (length
and internal diameter) but different in the particle size of the stationary phase.

Figure 2 Van Deemter plot

i) State van Deemter equation.
ii) Define one of the terms in van Deemter equation.
iii) Using the suitable term, discuss the effect of stationary phase particlesize on the efficiency of
chromatographic separation.

Q2 (15 marks)

a) Compounds A, B and C were separated using gas chromatography on a 20 m capillary column.

Table 1 tabulates the data obtained after the separation was completed.

Table 2

Compound tR (min) Width (min)

A 8.04 0.15
B 8.26 0.15
C 8.43 0.16

i) Calculate the number of theoretical plates for each compound.

2
 ii) Based on your answer in i), briefly explain why compounds A, B and C have different
numbers of theoretical plates.
iii) Calculate the resolution (Rs) for each pair of adjacent compounds.
iv) Suggest TWO factors that might improve the resolution between compounds B and C.

b) According to the van Deemter equation, column efficiency is significantly influenced by A, B and
C terms. Using suitable term(s), discuss why optimization of the flow rate is needed to improve
the efficiency of the column.

Q3 (25 marks)
a) A mixture of standard chlorinated pesticides with a boiling point range from 60°C–200 °C was
analysed using gas chromatography (GC). A 5 % polydimethylsiloxane (HP-5) column was used
and the column temperature was set isothermal at 100 °C. Nitrogen with a velocity of 30 cm/s was
utilised. Accurately, 1.0 μL of the sample was injected using a split ratio of 1:20.

i) Define isothermal conditions in GC separation.

ii) Briefly explain a suitable detector for this analysis.
iii) With justification, identify a suitable injection temperature for the sample.
iv) If the split ratio is changed to 1:100, predict the chromatogram obtained.
v) It was observed that two early eluting peaks were overlapped, while the last two peaks were
broad and took a long time to elute from the column. Analyse this observation.
vi) Recommend a suitable approach to improve the separation using appropriate examples.

b) In another study, a student conducted the separation of a hydrocarbon mixture containing

cyclohexane, cyclooctane and cycloheptane with the boiling point of 80.75C, 148.5 C and 110.5
C, respectively using GC with flame ionization detector (FID). The separation was done using a
polydimethylsiloxane (PDMS) column with an isothermal temperature of 150 C.
i) Briefly explain the suitability of the PDMS column in separating these hydrocarbons.
i) Describe the elution order of the hydrocarbon mixture.
ii) Sketch a chromatogram that could be obtained.
iii) Predict the separation of this hydrocarbon mixture if a 10 % phenyl PDMS column was used.

3
Q4 (25 marks)
a) Gas chromatography (GC) is an analytical technique used to separate and detect the chemical
components in a mixture. Figure 1 shows the important components of GC.

Figure 3 Components of GC

i) Label the components A, B, C and D.

A: __________________________________
B: __________________________________
C: __________________________________
D: __________________________________

ii) Explain briefly the function of A.

iii) State two factors to be considered in selecting C.

b) The analysis of four compounds; methane, 2-methylpropane, 2,2-dimethylpropane, and 2-

methylheptane (boiling point range between 130 °C to 200 °C) was conducted using gas
chromatography (GC). The separation was done using a phenyl polydimethylsiloxane column with
an isothermal temperature of 150 °C. The compounds were found to have retention times of 1.5,
1.8, 2.2 and 8.7 min, respectively, with resolution values of 1.0, 1.32, and 10.5.

i) From the results obtained, it can be concluded that an isothermal temperature of 150 C is not
efficiently separated all four compounds. Suggest a suitable temperature programming
approach to improve the separation.
4
 ii) Discuss your answer in ii).
iii) With justification, determine the appropriate temperature of the injector port to be set for this
analysis.

c) Captopril (Figure 4) is used to treat hypertension, congestive heart failure, and kidney problems
caused by diabetes, and to improve survival after a heart attack. In one study, wastewater containing
captopril was extracted using a solid phase extraction technique prior to gas chromatography
electron capture analysis.

Figure 4 Chemical structure of Captopril

i) Two types of GC columns, which are polyethylene glycol (PEG) and polydimethylsiloxane
(PDMS), are available in the laboratory. Choose which column is most suitable to separate
captopril.

ii) Justify the selection of column in ii).

iii) The GC-ECD is equipped with a split/splitless injection mode. Discuss the most suitable
injection mode for this analysis.

Q5 (29 marks)
a) Partition chromatography is a technique in which solutes are separated based on their partition
between a liquid mobile phase and a stationary phase.

i) Distinguish between normal-phase and reversed-phase chromatography.

ii) State one example of a typical stationary phase in reverse chromatography.
iii) In one study, a mixture containing n-hexanol and compound X was separated using a reversed-
phase partition HPLC. A mixture of methanol and water (30:70, v/v) was used as a mobile phase.
It was found that compound X eluted at a retention time of 20.9 minutes, while n-hexanol eluted
faster at a retention time of 5 minutes. Discuss briefly the finding.
iv) Based on the observation in iii), suggest a suitable gradient elution program for the mobile phase
to shorten the retention time of compound X.

5
 v) Analysis of cis and trans-pyrazoline was done using a polar column, polyethylene glycol (PEG).
However, only one peak was obtained. Explain.
vi) Select a suitable type of liquid chromatography and the mechanism involved in separating the cis
and trans-pyrazoline. Justify your answer.

b) Separation of chloride, fluoride and carbonate from a water sample can be performed using ion
exchange chromatography.

i) State the type of stationary phase and mobile phase required.

ii) Describe how the separation of those analytes is achieved.

c) A mixture containing five enzymes is separated using gel filtration chromatography (GFC): (1)
glutamate dehydrogenase (MW 290,000), (2) lactate dehydrogenase (MW 142,000), (3) enolase
(MW 67,000), (4) adenylate kinase (MW 32,000) and (5) cytochrome C (MW 12,400).

i) With a brief explanation, identify a column with a suitable Mw range that can successfully
separate all enzymes.
ii) If Bio-gel 150 with a molecular weight (Mw) range of 50,000-250,000 is used, illustrate the
expected chromatogram that could be obtained.

Q6 (30 marks)

a) HPLC instrument consists of guard and analytical columns.

i) Briefly describe the function of the guard column for the analysis of plasma samples.
ii) Typically, the stationary phase in an analytical column is silica gel. However, the usage of this
silica stationary phase is restricted to pH values between 2 and 8. Explain.

b) In partition chromatography, a mixture containing four aromatic hydrocarbons (n-propyl benzene,

benzene, phenol and toluene) was separated by HPLC at 254nm wavelength on an octadecyl (C18)
silica-bonded phase. The eluent used was 80 % methanol in water.

i) Arrange the order of elution of the aromatic hydrocarbons.

ii) Discuss the effect on the retention times of the compounds if the eluent was changed to 50 %
methanol in water.
iii) If the stationary phase was changed to a more polar column (bare silica), identify the elution
order of the aromatic hydrocarbons using 80 % methanol in water.
iv) Explain briefly why wavelength 254 nm was used for this separation instead of 280 nm.

6
c) A mixture of protein as listed in Table 3 was separated by Bio-gel 150 with a Mw range of 10,000-
90,000 Da.
Table 3

Protein Molecular weight (KDa)

Cystatine-C 95
Retinol 21
Ovalbumin 45
Galactosidase 116

i) Illustrate and label a chromatogram obtained for this separation.

ii) Recommend an approach to improve the separation in i), and estimate the order of elution.

d) A soil sample containing calcium, potassium and aluminium ions were collected from a local farm
and sent to the laboratory for analysis.
i) State suitable ion exchange column and suppressor column to separate all ions.
ii) Predict the order of elution of the mixture of calcium, potassium and aluminum ions Explain.

e) Two compounds (butane and iso-butane) were analysed by two types of HPLC); reversed-phase
partition chromatography and adsorption chromatography. Using appropriate justification
differentiate the chromatogram obtained from both separation techniques.