1.

0 INTRODUCTION

HPLC provide of reliable quantitative precision and accuracy for the determination of
the active ingredients and relate substances in the same run using a different solvent,
column and detectors that can be perform through the instruments. HPLC produce
excellent reproducibility and is able to use in a wide range of compound type. HPLC also
is the most suitable method for meeting most criteria quantitative analysis of a number
drug. Chromatography in include the method that having goal for the separation of
different type of components from a mixture based on their distributions capacity between
mobile phase and stationary phase. There may not be suitable method available for some
particular analyte in the specific sample matrix.

In this experiment, the sample is carried by a moving carrier gas stream which is
nitrogen. HPLC has ability to identify and separate the compound that present in any
sample that can be dissolve in liquid in trace concentration as low ppm. The retention
time of the sample will vary depend on interaction between the stationary phase,
molecules being analysed and the solvent used. As the sample pass through the column, it
will interact between the two phases at different rate. Analyte that has strong interaction
with stationary phase will elute later and the analyte that has less interaction at the
stationary phase will elute faster. The objective of this experiment is to optimize the
separation of mixture contain of 5 compounds using HPLC by change the composition of
mobile phase.
2.0 METHODOLOGY

a. Instrument set-up
Detector: 254 nm
Flow rate: 1.5 ml min-1
Mobile phase: acetonitrile:water

b. Effect of mobile phase on HPLC separation

The instrument was set up to use a mobile phase ration ACN:H2O (50:50 v:v) and the
sample was injected. After that, the mobile phase was change into composition 70:30.

c. Identification of components in the mixture

The compound was injected individually to identify the components of the mixture by
using the suitable HPLC conditions.

d. Separation using gradient elution

Base on the separation, the efficiency of the column was improved by gradient
elution.
e. Isocratic elution

ACN H2 O
50 50
70 30
3.0 RESULTS

A) Result of standard mixture run at ACN:H2O (50:50 v:v) of mobile phase composition.

PEAK RETENTION WIDTH,

TIME, min min
1 1.168 0.1713

2 1.357 0.1146

3 3.985 0.1719

4 6.914 0.2150

5 25.673 0.3153

Calculation of Resolution between peak 2&3.

2(1.357−1.168)
𝑅𝑠 = = 1.3221
0.1713+0.1146

B) Result of standard mixture run at ACN:H2O (70:30 v:v) of mobile phase composition.

PEAK RETENTION TIME,

min AVERAGE OF AVERAGE OF
1st run 2nd run TIME WIDTH, min
1 1.148 1.154 1.151 0.1922

2 1.272 1.264 1.268 0.1346

3 2.125 2.105 2.115 0.1078

4 2.853 2.824 2.8385 0.1255

5 6.210 6.114 6.162 0.16635

Calculation of Resolution between peak 2&3

2(2.115−1.268)
𝑅𝑠 = 0.1346+ 0.1078 = 6.988
C) Result of individual standard run at ACN:H2O (70:30 v:v) of mobile phase
composition.

NAME OF RETENTION TIME,

COMPONENTS min
Caffeine 1.142

Acetone 1.268

Methyl benzoate 2.101

Phenatole 2.838

Phenanthrene 6.248

D) The gradient elution condition to run the standard mixture.

HOLD TIME, COMPOSITION OF COMPOSITION OF

min ACETONITRILE WATER
0.1 10 90
0.5 10 90
1.0 50 50
1.3 60 40
1.8 70 30
2.3 10 90
E) Result of standard mixture that run by using gradient elution composition in the above
condition.

PEAK RETENTION
TIME, min
1 1.869

2 3.119

3 4.130

4 4.524

5 5.555
4.0 DISCUSSION

Isocratic is means constant composition which is the composition of mobile phase

remain constant throughout the analysis. In isocratic elution, the width of peak is increase
with retention time linearly according to equation of N which is the number of theoretical
plate. The late eluting peak will become broad and flat. Other than that, when the
composition of mobile phase is change during the separation process is described as a
gradient elution.
By gradient elution, the retention time will be decrease of the later eluting
components and will elute faster by producing narrow peak. This will improve the peak shape
for tailed peaks as the increasing the concentration of organic eluent pushed the tailing part of
a peak forward. This also will increase the peak height that is very important for trace
analysis.
In this experiment, the standard mixture was run at mobile phase composition of
ACN:H2O (50:50 v:v). Based on the chromatogram, the last peak was elute at 26.293min
which take a longer time to be eluted. So, we change the mobile phase composition to
ACN:H2O (70:30 v:v). The result show that the last peak eluted at 7.052 min which becomes
more faster than using ACN:H2O (50:50 v:v).
Hence, by using the condition of mobile phase composition at ACN:H2O (70:30 v:v),
the individual standard was run in order to identified the peak in the standard mixture. It was
found that caffeine was eluted at 1.142min followed by acetone at 1.268min, methyl benzoate
at 2.101min, phenatole at 2.838min, and phenanthrene at 6.248min.
After that, the standard mixture was run again by using gradient elution condition.
The aims was to separate the peak of compound 1 and peak of compound 2 and to decreased
the rate of time for compound to be eluted. Based on the result, we has successfully separated
peak of compound 1 and peak of compound 2 and also make the retention time becomes
more faster than using ACN:H2O (70:30 v:v) at isocratic elution condition.
 The hold time and the mobile phase composition used in gradient elution was shown
in the above result. It was found that the order of elution in standard mixture are caffeine,
acetone, methyl benzoate, phenatole and lastly phenanthrene. The retention times of
compounds are 1.869min, 3.119min, 4.130min, 4.524min, and 5.555min. Based on the result,
the aims of separation was achieved as it decreased the rate of time for compound to be
eluted.
Last but not least, in theoretically, the separation was based on the polarity of
compound. Since, we has used C18 column which is non-polar column, the mobile phase
must be polar solvent in order to achieved separation of compounds. At ACN:H2O (50:50
v:v), the polarity of solvent is more polar compared to ACN:H2O (70:30 v:v) which is less
polar. The less polar solvent will make the compound eluted faster. On the other hand, the
less non-polar compound (caffeine) will elutes first followed by the most non-polar
compound (phenanthrene). This is due to the principle of “like dissolve like” which most
non-polar compound will partition to the stationary phase while less non-polar compound
will partition to the mobile phase.
5.0 CONCLUSION

In conclusion, the separation of standard mixture by using ACN:H2O (70:30 v:v)

isocratic elution was the best compared to ACN:H2O (50:50 v:v). The individual standard
was run at ACN:H2O (70:30 v:v) using isocratic elution. Based on the result, caffeine was
eluted at 1.142min followed by acetone at 1.268min, methyl benzoate at 2.101min, phenatole
at 2.838min, and phenanthrene at 6.248min. After doing isocratic elution method, the
standard mixture was need to be run by using gradient elution method. We has successfully
separated peak of compound 1 and peak of compound 2 and also make the retention time
becomes more faster than using ACN:H2O (70:30 v:v) at isocratic elution condition. The aims
of experiment was achieved.

6.0 REFERENCES

1) High-performance liquid chromatography-Wikipedia (Retrieved on 11/5/2019)

https://en.m.wukipedia.org./wiki/High-performance_liquid_chromatoqraphy

2) High performance liquid chromatography (HPLC) | HiQ (Retrieved on 11/5.2019)

http://hiq.lindegas.com/en/analytical_methods/lquid_chromatography/high_performan
ce_liquid chromatography.html

3) High Performance Liquid Chromatography – Chemistry Libre Texts

https://chem.libretexts.org/Bookchelves/Analytical_Chemistry/Supplemental_Module
s_(Analytical_Chemistry)/Instrumental_Anaysis/Chromatography/High_Performance
_Liquid_Chromatography