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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
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.
2 1.357 0.1146
3 3.985 0.1719
4 6.914 0.2150
5 25.673 0.3153
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.
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.
Acetone 1.268
Phenatole 2.838
Phenanthrene 6.248
PEAK RETENTION
TIME, min
1 1.869
2 3.119
3 4.130
4 4.524
5 5.555
4.0 DISCUSSION
6.0 REFERENCES