(EXPERIMENT 2)

HIGH PERFORMANCE LIQUID

CHROMATOGRAPHY (HPLC), METHOD
DEVELOPMENT
NAME:

MOHAMAD NOR AMIRUL AZHAR BIN KAMIS

STUDENT ID:

2014647344

PARTNERS NAMES: 1.

MOHAMAD HAMIZAN BIN MOHD ISA

2.

MOHAMAD SHAFIQ BIN PARMAN

3.

MOHAMAD AZMIZAM BIN MOHAMAD

NOOR

DATE OF EXPERIMENT:

6/11/2014

DATE OF SUBMISSION:

8/12/2014

INTRODUCTION:
High Performance Liquid Chromatography is a type of separation analysis that uses
liquid mobile phase and liquid stationary phase. The separation occurs based on the
differences in the polarity of the analytes. The analyte that interacts most in the
stationary phase will elute later than the analyte that interact least in the stationary
phase. Reversed phase chromatography is used in which the stationary phase is
non-polar while the mobile phase is the polar mixture. The changes in the mobile
phase polarity will affect the interaction between the analyte with the stationary
phase and also affect the efficiency of the separation. Changes in the mobile phase
composition can be done either by isocratic elution whereas the composition of the
mobile phase is constant throughout analysis or by gradient elution in which the
composition of the mobile phase is change during separation either continuously or
in step in order to separate wide range of polarity of the analytes. The objective of
this experiment is to optimize a separation of caffeine, methyl benzoate, phenatole
and phenanthrene using HPLC by varying the mobile phase composition.

EXPERIMENTAL:
a. HPLC was set up with following condition:
Detector wavelength: 254nm
Mobile phase flow rate: 1.5mL/min
Mobile phase: acetonitrile:water
b. Effect of mobile phase on HPLC separation:
1. 20L of standard mixture was injected into the HPLC by using combination
of acetonitrile and water (50:50).
2. The ratio of acetonitrile to water was changed for the second injection to
70:30 (ACN:water).
3. The resolution of the two chromatograms was calculated and compared to
determine the best composition for that analysis.
c. Identification of components in the mixture:
1. Each of the standard compounds (caffeine, methyl benzoate, phenatole
and phenantrene) was injected with the optimized HPLC condition to be
compared with the retention time of the standard mixture.
d. Separation using gradient elution:
1. Gradient elution separation was performed based on the separation by
using isocratic elution to improve the efficiency of the column.

RESULT AND DISCUSSION:

A. Effects of the variation of composition of mobile phase on resolution (isocratic
elution):
* Calculation of resolution is based on peak 2 and 3 as references.
Composition of
mobile phase
(ACN:H2O)
50:50
70:30

Retention time of
peak 2 and 3 (min)
3.028, 5.170
1.645, 2.172

Base peak width

of peak 2 and 3
(min)
0.1581, 0.2412
0.0856, 0.1065

Resolution

22.30
5.49

B. Effects of the gradient elution program:

Composition of
mobile phase
(ACN:H2O)
75:25

Retention time of
peak 2 and 3 (min)
1.641, 2.139

Base peak width

of peak 2 and 3
(min)
0.0870, 0.1039

Resolution

5.22

C. The retention time of the standard mixture at optimized mobile phase

composition:
Standard compound
Caffeine
Methyl benzoate
Phenatole
phenanthrene

Retention time in
standard (min)
0.947
1.649
2.180
4.680

D. Sample calculation:
*70:30 (ACN:H2O) isocratic elution
Rs (2,3) =
= 5.49
*75:25 (ACN:H2O) gradient elution
Rs (2,3) =
= 5.22

Retention time in
mixture (70:30)(min)
0.942
1.645
2.172
4.637

Composition of the mobile phase is strongly affected the retention time of the
analytes to be eluted throughout the column. Since it is a reversed phase partition
liquid chromatography, the stationary phase is a polar while the mobile phase liquid
is non-polar. Strong eluent strength tends to decrease the retention time of the
analytes. Strong eluent strength is where the composition of the organic solvent
used in the ratio is higher than the water. In comparison between 50:50 (ACN:H 2O)
and 70:30 (ACN:H2O), 70:30 ratio has higher eluent strength so that the analytes
come out earlier than the analytes that used mobile phase composition of 50:50. The
separation quality is depending on the resolution between peaks that ideally 1.5.
Resolution that is higher than 1.5 will produce good separation between peaks but
needs longer retention time for the last analyte to be eluted. Low resolution than the
ideal value of 1.5 produces inadequate separation between peaks or in other words,
the peaks is overlap between them.
There is different is happening when the gradient elution mode is used. Gradient
elution mode is used when there are wide polarities of compounds to be separated.
Gradient elution changes the polarity of mobile phase composition throughout
analysis either continuously or in step during the separation. Gradient elution mode
will decrease the retention time for the analytes to be eluted throughout the column.
The gradient elution will give better separation, high efficiency and good resolution in
separating the mixture with wide polarities.
The qualitative analysis was done to identify the components in the mixture by
comparing the peaks in the mixture with the peaks of the standard compound. There
is caffeine indicated for the first peak followed by methyl benzoate, phenatole and
phenanthrene.

CONCLUSION:
The optimized mobile phase composition for the separation of the mixture is 70:30
(ACN:H2O). Higher composition of organic solvent will increase the solvent strength
that will shorten the analysis time. The first peak is corresponds to caffeine, secondly
is methyl benzoate, phenatole peak and lastly is corresponds to phenanthrene peak.

REFERENCES:
1. Norashikin S., Ruziyati T., Mardiana S. (2012), Analytical Separation
Methods Laboratory Guide (2nd edition), 3/10/2014.
2. Mardiana Saaid, Gas Chromatography Lecture Notes, 15/10/2014.