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TEST 2 June21 Q - fighting!

Analytical Separation Chemistry (MARA University of Technology (Shah Alam))

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ANALYTICAL SEPARATION METHODS June2021

UNIVERSITI TEKNOLOGI MARA

TEST 2 CHM510

NAME:……………………………………………………..STUDENT ID:………………………….

ANSWER ALL QUESTIONS (40 Marks)

QUESTION 1 (8 marks)

Answer TWO of the questions.

a) A strong solvent decreases the retention of analyte in HPLC. If the separation was
done using a reversed-phase chromatography, which mobile phase composition is
stronger, 40/60 methanol/water or 50/50 methanol/water? Explain your selection.
(4 marks)
For a reverse phase chromatography, the stationary phase is non polar
Have a strong interaction with he non polar analyte
Strong solvent has a lower polarity make the non polar analyte eluted out faster
The mobile phase composition of 50/50 metahnol/water is stronger

b) Two compounds, A and B were injected onto a chromatographic column. A has a

greater affinity for the column packing than does B. Compound B is more soluble in the
mobile phase than is A. Which compound will be swept through the column more
quickly? Justify your choice.
(4 marks)
Compound b

Compound A has greater affinity for the column packing indicate A has a stronger
interaction with the stationary phase
And will retained for longer time
While compound b more soluble in the mobilephase. It means the compound B spends
more time in the mobile phase than compound A, thus B will move further along the column
than A

c) Explain the reason why a guard column is used in HPLC but not in GC systems.

(4 marks)
The mbile phase in HPLC is solvents, while the mobile phase in the Gc system
using gases(1) and is inert(1) . thus the guard is rquired in HPLC to remove
the impurities and suspended solids from reaching the analytical column(1)as
well as to protect and increases the lifetime of the analytical column (1)

QUESTION 2 (12 marks)

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ANALYTICAL SEPARATION METHODS June2021

a) A mixture of acetone, acetamide, and 1,2-dichloroethane were separated with a C18

column using a reversed-phase Liquid Chromatography system. Given are the polarity
of solvent in increasing order: 1,2-dichloroethane < acetone < acetamide.

i) Suggest a suitable mobile phase solvent/solvent system that can be used for the
analysis of this mixture.
(1 mark)
Any polar solvent or mixture of those : water , methanol, acetonitrite, etc

ii) If the C18 column is replaced by a column with –C 2H4CN functional group attached
to the siloxane backbone and n-hexane as the mobile phase, sketch a
chromatogram (label the peaks) that would be obtained.
(3 marks)

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ANALYTICAL SEPARATION METHODS (CHM510) Test2_Jun2021

b) A mixture of five pharmaceutical test compounds (and three impurity substances) was
analysed using high-performance liquid chromatography (HPLC) on a 25 cm C18
column (5 µm particle size). The mobile phase consisted of a mixture of 40%
Acetonitrile and 60% water. A flow rate of 0.8 mL/min was used.

i) As shown in Figure 1a, all peaks were eluted very slowly and the last two
peaks were not completely resolved. Give one reason and suggest one
approach to overcome the problem.
Elute slowly because the interaction between analytes and the stationary
phase is too strong (1) while the slvents are not strong enough to desorb the
analytes (1) the last two peaks not completely resolved due to their polarities
are about the same.

Increase the solvent strength / change the c18 to a less retentive column like
c8 c4 phenyl
(4 marks)

Time (min)

Figure 1a

4) The separation was improved by doing some modification in an analytical

method as shown in Figure 1b. Suggest a suitable method development to obtain
this chromatogram.
(4 marks)
Start with a stronger solvents system by lowering the polarity of solvent(1) such
as using mobile phase composition with 60 acetonitrile and 40 water (1) and then
increae it from 60 to 90 acetonitrile (1) the elution can be futher faster by using a
hingher flowrate such as 1.2 ml/min (1)

Time (min)

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ANALYTICAL SEPARATION METHODS (CHM510) Test2_Jun2021

Figure 1b

QUESTION 3 (12 marks)

a) A mixture of proteins as listed in Table 1 was separated on Size Exclusion

Chromatography (SEC). The exclusion range of the gel was 25,000 Da to 120,000 Da.

Table 1

Protein Molecular weight

Urease 482,700
Catalase 242,500
Lactoglobin 37,100
Hemoglobin 64,500

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

(3 marks)

1, catalase and urease

2. haemoglobin
3. lactoglobin

ii) In your opinion, is this a good method to purify urease? Justify your answer.
(3 marks)
No.
Catalase and urase above the exclusion limit therefore too large to be trapped in the
stationary phase (1) and elute together without separation

b) Ion-exchange chromatography (IEC) is used to purify the water. For water softening,
this is usually done with sulfonated polystyrene beds that are supersaturated with
sodium to cover the bed surface. As water passes through this resin bed, ions attach
to the resin beads releasing the loosely held solution into the water as shown in Figure
2.

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ANALYTICAL SEPARATION METHODS (CHM510) Test2_Jun2021

Figure 2: Softening Process

Source: SKIPTON (2008)

i) Predict and justify the elution order of analytes for the above separation.
(3 marks)
Na+ mg2+ ca2+
ii) Suggest a stationary phase that can be used to separate each of the above
sample mixtures.
(1 mark)

Carboxylic acid
iii) Give one of the reactions that occur in the separation between the analyte and
the stationary phase mentioned in (ii). (Choose one analyte as an example)
(2 marks)

QUESTION 4 (8 marks)

Consider the separation of five benzene compounds:

n-ethylbenzene, n-propylbenzene, 2-chlorophenol, 1-chlorophenol and benzamide.

Column: Allshere Cyno(polar) , silica 250 mm x 4.6 mm x 5 m
Mobile phase: (A) water, (B) methanol
Gradient: (Time, %B), (0,20), (2,20), (15,80), (18,80)
Flow rate : 1 mL/min
Detector: UV, 254 nm

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ANALYTICAL SEPARATION METHODS (CHM510) Test2_Jun2021

a) Explain why only three peaks were displayed in the chromatogram. Suggest the
appropriate LC technique to overcome the above separation.
(3 marks)
1- Cholorphenol and 2 cholophenol are isomer that have different steric structure
hence different physiosorption characteristic

Normal phase partition separation is based on differences in polarity , therefore

unable to separate isomers of the same polarity

Adsorption chromatography

3) Propose a better gradient approach that may shorten the analysis time (improve
the resolution) of the last two peaks. Justify your answer.
(3 marks)
Start with low solvent strength (water:methanol: 80:20) to separate the first 3
compounds (1) . then, increase the solvent strength of the final composition up to
10:90 (1) at 10 min to shorten the retention of the last 2 compunds

c) Of the above compounds, which one would you expect to elute last from the column.
Justify your answer.
(2 marks)

Benzamide

The stationary phase of allshere cyano is polar. Thus the more polar compound has
a stronger interaction with the stationary phase and is retained longer

END OF QUESTION PAPER

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