1.

0 INTRODUCTION
High performance liquid chromatography is basically a highly improved form of
column chromatography. Instead of a solvent being allowed to drip through a column under
gravity, it is forced through under high pressures of up to 400 atmospheres. That makes it
much faster.
It also allows to use a very much smaller particle size for the column packing material
which gives a much greater surface area for interactions between the stationary phase and
the molecules flowing past it. This allows a much better separation of the components of the
mixture.
The other major improvement over column chromatography concerns the detection
methods which can be used. These methods are highly automated and extremely sensitive.
Typical HPLC system consists of followings (Figure 1).

Figure 1 Components of HLPC system

In this experiment, 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
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 to separate wide range of polarity of
the analytes.
The objective of this experiment is set to optimize a separation of caffeine, methyl
benzoate, phenatole and phenanthrene using HPLC by varying the mobile phase composition.
2.0 METHODOLOGY
The instrument was set up to 245 nm with flow rate of 1.5 mL min -1. The mobile
phase used is acetonitrile and water. Ratio of acetonitrile : water (50:50 v:v) was set up to use
as a mobile phase and the sample is injected. Then, the mobile phase composition is changed
to 70:30 in ratio. After that, the best composition of mobile is chosen. Each component was
then injected individually to identify the component contains in the mixture using the
selected HPLC conditions. Based on the separation, a gradient elution separation is performed
to improve the efficiency of the column.
3.0 RESULTS AND CALCULATIONS

a) Effect of the mobile phase on HPLC separation (isocratic elution)

Composition of mobile phase (ACN: H2O) 50 : 50

Retention time Base peak Resolution,

Injection Peak
(min) width (min) Rs

1.174, 0.1211,
1 and 2 1.68
1.376 0.1189
4.079, 0.1189,
2 and 3 17.99
1.376 0.1816
1
7.064, 0.1816,
3 and 4 13.77
4.079 0.2519
26.479, 0.8178,
4 and 5 36.30
7.064 0.2519

Composition of mobile phase (ACN: H2O) 70 : 30

Retention Base peak Resolution, Average

Injection Peak
time (min) width (min) Rs resolution, Rs

1.242, 0.0977,
1 0.82
1.151 0.1242
1 and 2 0.86
1.251, 0.0973,
2 0.89
1.151 0.1270
2.132, 2.137,
1 7.59
1.242 1.251
2 and 3 7.58
0.1368, 0.1367,
2 7.57
0.977 0.0973
2.864, 0.1694,
1 4.78
2.132 0.1368
3 and 4 4.80
2.872, 0.1690,
2 4.81
2.137 0.1367
6.393, 0.4076,
1 12.23
2.864 0.1694
4 and 5 12.25
6.403, 0.4068,
2 12.27
2.872 0.1690
b) Effect of the gradient elution

Time (min) ACN: H2O

0-1.4 40:60
2.4-2.5 75:25
2.5 - end 85:15

Retention Base peak

Resolution, Rs
time (min) width (min)
1.161, 0.1079,
Peak 1&2 1.32
1.292 0.0907
1.292, 0.0907,
Peak 2&3 12.73
2.691 0.1291
2.691, 0.1291,
Peak 3&4 5.34
3.339 0.1136
3.339, 0.1136,
Peak 4&5 10.24
4.976 0.2061

c) The identification of the compounds in the mixture

Retention time in Retention time in individual

Standard compound individual standard mixture (70:30 ACN:H2O)
(min) (min)
Acetone 1.291 1.251
Caffeine 1.155 1.151
Methyl benzoate 2.143 2.137
Phenanthrene 6.426 6.403
Phenetole 2.878 2.872
d) SAMPLE OF CALCULATION

2( tR2 − tR1 )
Resolution, Rs = 𝑊𝑏1+𝑊𝑏2

Isocratic elution , (70:30 ACN:H2O)

2(1.251−1.151)
Rs(12) =
0.1270+0.0973

= 0.90

Gradient elution, (40:60 ACN:H2O)

2(1.292−1.161)
Rs(12) =
1.1029+0.0907

= 1.32
4.0 DISCUSSION

In this experiment, method development of High Performance Liquid

Chromatography (HPLC) been used to optimized the separation of 5 compound
mixture which is caffeine, acetone, methyl benzoate, phenatole and phenanthrene
using HPLC by varying the mobile composition. This technique has same operational
basis as liquid chromatography however, this technique provides far better separation
of component of the mixture.
At the beginning of this experiment, the mobile phase ratio was set up to
50% of acetonitrile and 50% of water. After all the standard sample which are caffeine,
acetone, methyl benzoate, phenatole and phenanthrene were injected, the ratio was
changed to 70:30. At 50:50 ratio, peak 1 and peak 2 did not separate well but for the
next two peaks, it comes out nicely. At 70:30 ratio, peak 1 and 2 also did not separated
well followed by peak 3,4 and 5 that separated well. But the retention time for the
fifth peak is very long.
The peak might not have separated well because of sample did not
degassing properly that cause air bubble in the mobile phase that fluctuant the
instrument. Moreover, the instrument should be purging to let the pressure stable.
In this experiment we also determine the components of the mixture by
comparing with the peak of the standard compound. Firstly, the compound is caffeine
with retention time of standard 1.151 min followed by acetone at 1.251 min, methyl
benzoate 2.137 min, phenetole at 2.872 min and lastly phenanthrene with 6.403 min.
The result of the experiment can be improved by adjusting the gradient elution
programming to reduce the overlapping of peak 1 and peak 2, produce a better shape
of peak for peak 3 and shorten the analysis time for peak 5 so that it not retained too
long in the column.
5.0 CONCLUSION
The optimized mobile phase composition of this experiment is 70:30(ACN:H2O).
The higher the composition of the organic solvent which is ACN the more increase the
solvent strength lower the polarity of the solvent that will shorten the analysis time.

The peak order is caffeine followed by acetone, methyl benzoate, phenatole and
phenanthrene.
6.0 REFERENCES

ANALYTICAL SEPARATION METHODS LABORATORY GUIDE, 2nd edition.

Nor’ashikin Saim, Ruziyati Tajuddin, Mardiana Saaid. Penerbit Press Universiti Teknologi
MARA.

HPLC Lab Report Discussion. (n.d). Retrieved from

https://www.coursehero.com/file/14226088/exp-hplc-discussion-sahaje/

SHOWA DENKO K.K. ( n.d) Retrieved from

https://www.shodex.com/en/kouza/a.html

Experiment 2 HPLC CHM510. (n.d). Retrieved from

https://www.scribd.com/doc/315267522/Experiment-2-Hplc-chm510