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TEST 2 December 2020 Q - fighting!

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

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TEST 2:CHM510_Dec2020

UNIVERSITI TEKNOLOGI MARA

CHM510: ANALYTICAL SEPARATION METHODS
TEST 2: HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

NAME:_____________________________________ STUDENT NO:_______________

ANSWER ALL QUESTIONS (40 marks)

QUESTION 1
A water sample consists of five benzene compounds (n-methylbenzene, n-propylbenzene, n-
butylbenzene, 2-chlorobenzylalcohol and 3-chlorobenzylalcohol) was analysed using
reversed phase HPLC under the following conditions:
Column: Octadecyl bonded phase column (150 x 4.6mm x 10µm)
Mobile phase: A: H2O B: methanol
Gradient: (Time, %B), (0, 40), (4, 40), (12, 80), (14, 90)
Flow rate: 1.5 mL/min
Detector: UV at 254nm
The separation was completed in 18 minutes as shown in Figure 1.

4 8 12 16 20
Reten琀椀on 琀椀me, min.

Figure 1: Separation of benzene compounds

a) Explain why a UV detector with 254nm is selected in this experiment.

It would detect anything with the benzene ring and it will detect any pi to pi transitioned
(2 marks)

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TEST 2:CHM510_Dec2020

b) Describe the separation and the elution order of the above benzene compounds.

(6 marks)
C18 is non polar column
Non polar interact more and retain longer in the column
More polar eluted first due to less interaction
c) Discuss the effect of the above separation if:
i) The octadecyl bonded phase column was changed to an octyl bonded phase
column.

(4 marks)
C18 to c8
C8 less non polar than c18
Elution order still the same
Number of carbons reduce
Retention time reduce
ii) The gradient elution was changed to isocratic elution with 60:40 (H2O:methanol).

(2 marks)
First two peak Overlapped with the same retention time
Other three peaks well separated but longer retention time

d) Explain why compounds 1 and 2 were not separated. Suggest and discuss another
alternative HPLC method that can be used to separate them.

(5 marks)
Because it types of isomers

Used adsorption chromatography

Main advantages to separated isomers(cis and trans) have very different
physio sorption characteristics due to steric effect in the molecules
QUESTION 2
A mixture of five sugars is to be separated by Size Exclusion Chromatography (SEC) using
the columns given in Table 1.
Table 1

Column Pore Size (Å) Molecular Weight Range (kDa)

Unix SEC-300 300 0.1-2.0
Unix SEC-400 400 0.5-2.0

If the five sugars to be separated containing molecular weights as tabulated in Table 2:

Table 2

Sugar Molecular weight (kDa)

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TEST 2:CHM510_Dec2020

Xylose 0.150
Glucose 0.180
Maltose 0.342
Maltoheptose 1.153
Starch 1.500

a) Discuss which one of the columns will be able to separate all five sugars.
(6 marks)

Unix sec-300
Because the molecular weight of the largest analyte starch is 1.5 and the molecular
weight of the smallest analyte xylose is 0.150
Unix sec-300 is able to separate all the analytes because the molecular weight range
is between 0.1 and 2.0
Will be separated as 5 individual peaks
b) Draw and label the expected chromatogram that will be obtained in the separation
based on your answer in (a).
(2 marks)

c) Suggest with reasons, a way to separate all the sugars into five individual peaks for
the unchosen column based on your answer in (a).
(3 marks)
Use a column that has total permeation limit must be smaller than 0.150 kda
Total exclusion limit must be maintain 2.0 kda
All sugar will be within the permeation region and will separated to individuals peaks
QUESTION 3
Three ions which are nitrate, hydrogen sulphate, and dihydrogen phosphate ions in water
samples are determined on a single filtered and unacidified sample with ion-exchange liquid
chromatography.
a) Suggest the type of ion exchange liquid chromatography and resin that can separate
the ions in water samples. Justify your choice.
(3 marks)
Anion exchange chromatography because there ions are anions.
The resin that used to separated is support-N(ch2)3*OH- or primary amine.
b) Write the reaction that occurs in the analytical column that has been chosen based
on your answer in (a). Choose one of the ions as your example.
(2 marks)
No3- + support-N(ch2)3 -OH OH - + support-N(ch2)3 -No3-

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c) In the analysis above, most of the ions could not be detected due to the low
sensitivity of the conductivity detector. Discuss the reason for the mentioned problem
and suggest an approach to overcome the problem.
(5 marks)
Mobile phase contains ion than Create background conductivity make it difficult to
measure the conductivity due to only the analyte ions as they exit the coloum

Used suppressor column

Column packed with second ion exchange resin of opposite charge to the analytical
column and placed after the analytical column

END OF TEST QUESTIONS

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