CHM510 HPLC Assignment (total marks= 65)

Sample problem 9
A mixture of 4 relatively non polar drugs was analyzed on a 25 cm C18 column (3 µm particle size). The
mobile phase contained a mixture of 40% MeOH & 60% water. A flow rate
of 0.8 mL/min was used. The column dead volume was measured to be 1.5 mL/min.
Calculate the elution time (in min) of a non-retained solute.

Sample problem 10
A mixture of 4 relatively non polar drugs was analyzed on a 25 cm C18 column (3 µm particle size). The
mobile phase contained a mixture of 40% MeOH & 60% water. A flow rate of 0.8 mL/min was used. The
column dead volume was measured to be 1.5 mL/min.

i. 2 of the drugs in the mixture were found to have very long retention time. If you wished to
speed up the elution of the 2 drugs without affecting the elution of the earlier peaks, suggest a
suitable gradient approach for the separation. Explain.
(6 marks)
To speed up the retention time of later eluting peaks, the ratio of mobile phase composition
must be change to higher organic solvent content. Organic solvent will reduce the retarding
strength of the aqueous component. However, to avoid the disturbance to early eluting peak,
the sample must be injected while weaker mobile phase is being applied to the system. The
strength of mobile phase is later increase from 40% of MeOH to 70% MeOH while water is
decrease from 60% to 30%. Hence, the retention time of later eluting components can be
decrease as well as give narrow peaks.

ii. Briefly explain what would happen to the tR of the compounds if a 25 cm C8 column (3 µm
packing) was used instead of the C18 column without changing other conditions.

(4 marks)
C18 column has octadecylsilane as its stationary phase. Therefore, it will show higher retention
time as the analytes elutes slowly in this column. Nonpolar compound also moved down the
column slowly. On the other hand, C8 column has octylsilane as its stationary phase.
Therefore, it will show low retention time as the analytes elutes faster in this column. Nonpolar
compound moved down the column rapidly.

Sample problem 11
When using a C18 stationary phase is it more beneficial to increase or decrease mobile phase composition
polarity during elution? Explain.
(5 marks)
C18 stationary phase is nonpolar, hence it will highly interact with nonpolar compounds. Therefore,
nonpolar compound will be retained longer in the column. However, the polar compound will be the earliest
to be eluted. In fact, there are possibility that the polar compound did not interact with the stationary phase.
Therefore, the polarity of the mobile phase needs to be decrease to make it slightly nonpolar. Hence, the
affinity of hydrophobic analyte with hydrophobic stationary phase will be weaker. Therefore, the retention
time can be decrease by adding more organic solvent to the eluent.

Sample problem 12
A mixture of alcohol compounds was analyzed on a 25 cm C18 column. The mobile phase contained a
mixture of 30% MeOH & 70% water. Explain how the retention would be affected if
the composition of the mobile phase was changed to 70% MeOH & 30% water.
(5 marks)
The retention time of the composition will be decrease. As more organic solvent is added to mobile phase,
the polarity of mobile phase will be slightly nonpolar. Organic solvent also reduces the retarding strength of
aqueous component. Hence, the interaction of nonpolar compound with nonpolar stationary phase will be
weaker. Therefore, the strongly retained compound can be eluted faster as the interaction has become
weaker. Retention time also will be decrease.

Sample problem 13
Discuss the reason of the mentioned problem & suggest an approach to overcome the problem.

In a separation using a C18 column with a mobile phase of ACN/water : 80/20, the first 2 peaks eluted very
close to tM.
(6 marks)
C18 column has nonpolar stationary phase. Therefore, when the mobile phase composition is
highly nonpolar, the early two peak will be very close to tM because strong solvent allows analytes
to be rapidly elutes from the column. The interaction of polar compound with stationary phase

Sample problem 14
A test mixture consisting of benzene, methylbenzene, phenol & acetophenone is to be separated on a
C18column with a mobile phase of 50/50 MeOH/water.

i. Describe the order of elution for these compounds.

ii. How would you change the composition of the mobile phase so as to reduce the retention of
the compounds?
(4 marks)
To reduce the retention time of the compound, the polarity of mobile phase needs to be
increased. The higher the polarity of mobile phase, the faster the elution time.

iii. If the C18 bonded phase column contained unreacted silanol groups, how would the tR be
affected? Briefly explain the process to reduce the concentration of unreacted silanols.
(5 marks)
Retention time will be longer. The concentration of unreacted silanols can be reduced by end-
capping process. The unreacted silanols is end-capped by adding silylating agent such as
trimethylchlorosilane. Silylating agent will bond with residual silanols groups on packing
surface. It also can cut down on undesirable adsorption as unreacted silanol group are capable
of adsorbing polar molecules,

Sample problem 15
Give a reason for the mentioned problem & suggest an approach to overcome the problem.
In normal phase separation of 5 comps using isocratic mixture of hexane:acetone (80:20), the retention
time for the last 2 peaks were too long.
(5 marks)

Sample problem 16

Give a reason for the mentioned problem & suggest an approach to overcome the problem.

ii) A gradient elution for a normal phase separation of a series of comps with a wide range of polarities
starts with 80/20 hexane/acetone & ends with 50/50 hexane/acetone.
(5 marks)

Sample problem 17
In a gradient elution normal phase separation, what should be changed in regard to acetone (starting
solvent) & hexane when the following situation develop?

i. The peaks are bunched together near tM.

Sample problem18
i) On a normal phase LC column, a compound had a tR of 15 min. Which solvent, hexane
or diethyl ether would increase the tR? Explain why.
(5 marks)

Sample problem19
ii) On a normal phase LC column predict the effect on the tR of comps 1(Anthracene (3
benzene ring fused structure)) & comp 3(Benzene) if the mobile phase is changed from 80/20
hexane/acetone to 50/50 hexane/acetone.
(5 marks)