COURSE CODE: CHM510

NAME: NURUL HUSNA BINTI IBRAHIM

NO MATRIC: 2018226626
TITLE EXPERIMENT: EXPERIMENT 2: HIGH PERFORMANCE
LIQUID CHROMATOGRAPHY(HPLC) METHOD
DEVELOPMENT
LECTURER’S NAME: MADAM ZARILA
LABORATORY PARTNER’S NAME:
1. HUSNA AFIFAH BINTI RAZALI (2018801414)
2. NUR AMIRAH BINTI AHMAD (2018801966)
3. FATIMAH AZZAHRAH BINTI MOHD FAIRUZ
(2018414328)
INTRODUCTION
High Performance Liquid Chromatography (HPLC) is a chromatographic method that is used
to separate mixture of compounds to identify or purify individual components of the mixture.
It is suitable for separating non-volatile species or thermally fragile ones. HPLC is divided
into two phase which is normal-phase chromatography and reverse-phase chromatography.
In this experiment, we used the reverse-phase chromatography which the stationary phase is
non-polar while the mobile phase is polar. We use acetonitrile: water as our mobile phase and
C18 column as our stationary phase. By increasing the mobile phase polarity will increase the
elution time and the most polar component will elute first. Therefore, changes in polarity of
the mobile phase can affect the interactions of the sample and stationary phase and thus
changes the efficiency of HPLC separation. In order to changes the mobile phases can be
done either by isocratic elution or gradient elution.
In this experiment, we use isocratic elution in order to identify the optimized mobile phase
composition to inject the individual standard in the gradient elution. Therefore, we have to
decide between 50:50 ratio or 70:30 ratio is the best for HPLC separation. Gradient elution is
composition of the mobile phase is changed during the separation.

OBJECTIVE
The objective of this experiment is to optimize the separation of a mixture of five compounds
(caffeine, acetone, methyl benzoate, phenatole and phenanthrene) using HPLC by varying the
mobile phase composition.

REAGENTS AND SOLUTIONS

a. HPLC grade acetonitrile
b. Deionised water
c. Standard mixture of caffeine, acetone, methyl benzoate, phenatole and phenanthrene
(~100 ppm)
INSTRUMENTS
Liquid chromatograph (Agilent G1314A HPLC) equipped with diode array detector (DAD),
RP C18 column and 20µL sample loop.
PROCEDURE
a. Instrument set up (may vary depending on instrument)
Detector wavelength: 254 nm
Flow rate: 1.5µL min-1
Mobile phase: acetonitrile: water

b. Effect of mobile phase on HPLC separation

The instrument is set to use a mobile phase ratio of acetonitrile: water (50:50) and the
standard mixture is injected. Then, the mobile phase composition is changed to 70:30.

c. Identification of components in the mixture

Each compound is injected individually to identify the components of the mixture by
using the optimum mobile phase composition that have been decide earlier.

d. Separation using gradient elution

A gradient elution is performed based on the separation above to improve the
efficiency of the column.
DATA AND RESULTS
R s=2 ¿ ¿

(A) ISOCRATIC STANDARD MIXTURE 50:50

Injection Peak Retention time Width (min) Resolution, RS
(min)

1 1.178 0.1517 R S12=1.2795

R S23=15.4629

2 1.368 0.1453 R S34=13.5301

R S45=37.9837
1
3 3.907 0.1831

4 6.726 0.2336
 5 24.396 0.6968

ISOCRATIC STANDARD MIXTURE 70:30

Injection Peak Retention time Width (min) Resolution, Rs Average
(min) resolution

1 1.156 0.1237
R S12=0.9485
2 1.272 0.1209 R S23=6.0075
R S34=4.6736
1 3 2.077 0.1471 R S45=37.9837

4 2.775 0.1516

R S12=0.9453
5 5.996 0.2131 R S23=5.9551
R S34=4.1139
1 1.157 0.1278 R S45=29.1935

2 1.274 0.1206 R S12=0.9420

R S23=5.9026
R S34=3.5541
3 2.080 0.1525
2 R S45=20.4032

4 2.571 0.1238

5 6.012 0.2135
 (B) GRADIENT ELUTION

Injection 1

Time (min) ACN: H2O

0-1.8 50:50
1.8-3.0 70:30
3.0-8.0 85:15

Injection 2

Time (min) ACN: H2O

0-1.8 50:50
1.8-8.0 70:30

Retention Resolution, Average

Injection Peak Width (min)
time (min) Rs resolution, Rs
1.136 0.1598 R S12=¿1.0877
1
1.278 0.1013
1 and 2 R S12=¿1.0970
1.138 0.1540 R S12=¿1.1063
2
1.278 0.0991
1.278 0.1013 R S23=¿8.0618
1
2.582 0.2222
2 and 3 R S23=¿8.4890
1.278 0.0991 R S23=¿8.9162
2
2.582 0.1934
2.582 0.2222 R S34=¿4.8646
1
3.489 0.1507
3 and 4 R S34=¿5.0517
2.582 0.1934 R S34=¿5.2388
2
3.487 0.1521
3.489 0.1507 R S45=¿8.9502
1
5.403 0.2770
4 and 5 R S45=¿8.9528
3.487 0.1521 R S45=¿8.9554
2
5.403 0.2758
 (C) Identification of compound in the mixture

Standard Compound Retention time in Retention time in

individual standard standard mixture (70:30
(min) ACN: H2O (min)
Caffeine 1.154 1.156
Acetone 1.289 1.272
Methyl Benzoate 2.085 2.077
Phenatole 2.789 2.775
Phenanthrene 6.033 5.996

DISCUSSION
Objectives of this experiment is to optimize the separation of a mixture of five compounds
which is caffeine, acetone, methyl benzoate, phenatole and phenanthrene by varying the
mobile phase composition. Therefore, we use two method for the separation which is
isocratic elution and gradient elution. Isocratic elution is used in order to determine the
optimum ratio for mobile phase composition in order to inject the standard individual mixture
later. Gradient elution is used for improving the efficiency of column by ramping the ratio in
order to get better retention time in the isocratic elution result.
In the isocratic elution (50:50 ratio) we can see that the resolution between peak 1 and peak 2
is 1.2795 which is close to the ideal resolution that range between 1.5-2.0 and the peak has
well separated peaks compared to other peaks. While in isocratic elution (70:30) the
separation can be considered as a very good peaks because the resolution value is more than
1.5 and the peaks shown are very sharp even though peak 1 and peak 2 is overlapped.
However, the separation is still not efficient enough as we need to improve the retention time
and also the resolution. Therefore, we need to use gradient elution method in order to get
better retention time and resolution. We need to change the composition ratio along with the
time in order to get sharp and well separated peaks. Therefore, in the first 1.8 minutes we
used 50:50 ratio composition in order to avoid the overlapping in the peak 1 and 2. Then in
the next 1 minutes, we use 70:30 ratio to get a better resolution and to produce sharp peaks
and lastly we used 85:15 ratio in order to have a faster retention time than the standard
isocratic elution mixture.
Thus, we can use the gradient elution in order to optimized the separation of the mixture of
five compounds which we can use ramping ratio method where we changed the ratio
throughout the time. We can conclude that from 5 individual standard mixture, caffeine
eluted first followed by acetone, methyl benzoate, phenatole and lastly phenanthrene.

CONCLUSION
The optimized composition of the mobile phase for mixture separation is 70:30 (acetonitrile:
water). Caffeine eluted first, followed by acetone, methyl benzoate, phenatole and
phenanthrene.
REFERENCES
1. Hoefler, A. C., & Coggon, P. (1976). Reversed-phase high-performance liquid
chromatography of tea constituents. Journal of Chromatography A, 129, 460-463.

2. Heydari, R., & Mousavi, M. (2016). Simultaneous determination of saccharine,

caffeine, salicylic acid and benzoic acid in different matrixes by salt and air-assisted
homogeneous liquid-liquid extraction and high-performance liquid
chromatography. Journal of the Chilean Chemical Society, 61(3), 3090-3094.

3. https://chem.libretexts.org/Courses/University_of_California_Davis/UCD_Chem_115
_Lab_Manual/Lab_2%3A_High_Performance_Liquid_Chromatography

4. http://fareastzareul1994.blogspot.com/2018/04/experiment-2-high-performance-
liquid.html