UITM ARAU PERLIS

Experiment 2

High Performance Liquid Chromatography (HPLC) : Method Development

Name : Nur Izzati Binti Othman Basri

Group : RAS2452C
Matrix No. : 2020991379
Date Of Experiment : 5 May 2020
Date of Submission : 12 May 2020
Lecturer’s Name : En Mohd Lias
Objectives

To optimise the separation of a mixture of five compounds (caffiene, acetone, methyl

benzoate, phenatole and phenanthrene) using HPLC by varying the mobile phase
composition.

Abstract

The separation dependent upon interaction of the analytes with mobile phase and
stationary phase. Analysis of mixtures of non-volatile compound. The composition of
mobile phase in isocratic elution and gradient elution which contribute to the efficient
separation of mixture of five compound are examined. The elution rate of a compound
depends on the polarity. This experiment is examined high performance liquid
chromatography, including the interaction of mobile phase and stationary phase due to
the difference in polarities of the analytes and composition of mobile phase in
different type of elution that can changed the efficiency of HPLC separation. The
concepts of retention time and resolution is used for five compounds which were
caffiene, acetone, methyl benzoate, phenatole and phenanthrene. At the end of the
experiment,the resolution (Rs) is measured to know of how well species are separated.

Introduction
The fundamental basis for HPLC consists of passing a sample (analyte mixture) in a
high pressure solvent (mobile phase) through a column packed with sorbents
(stationary phase). The mobile phase acts as a carrier for the sample solution. As the
analytes pass through the column, they interact between the two phases, mobile and
stationary, at different rates. The difference in rates is primarily due to the difference
in polarities of the analytes. The analytes that have the least amount of interaction
with the stationary phase or the most amount of interaction with the mobile phase will
exit the column first. Therefore, changes in the polarity of the mobile phase can affect
the interactions of the sample and the stationary phase and thus changes the efficiency
of your HPLC separation. Changes in mobile phase can be done either by isocratic
elution or gradient elution.
Isocratic elution
The mobile phase composition remains unaltered during the separation. The mobile
phase may comprise of a single solvent or pre-mixed mixture of solvents.
Gradient elution
The composition of the mobile phase is changed during the separation. Two or more
solvents that differ in polarity are employed. After sample introduction, the ratio of
these solvents is programmed to vary either continuously or in steps.

Reagents and Solutions

a. HPLC grade acetonitrile
b. Deionised water
c. Standard mixture of caffiene, acetone, methyl benzoate, phenatole and
phenanthrene (~100 ppm)

Instrument
Liquid Chromatography (Agilent G1314A HPLC) equipped with diode array detector
(DAD), RPC18 column and 20 µl sample loop.

Analytical Procedure
a. Instrument set-up (may vary depending on instrument):
Detector wavelength : 254 nm
Flow rate : 1.5 µl/min
Mobile Phase : acetonitrile water

b. Effect of mobile phase on HPLC separation

Mobile phase ratio of acetonitrile:water (50:50) was set up and the standard
mixture is injected. Then, changed the mobile phase composition to 70:30.

c. Identification of components in mixture

To identify the components of the mixture each compound is injected
individually using the selected mobile phase composition.

d. Separation using gradient elution

For the gradient elution to do the separation, need to improvise the ratio of the
column by conducting the gradient elution method.
Results
Table 1 : The consequences of the change composition of mobile phase on resolution
by isocratic elution
Table 2 : The impact of composition of the mobile phase by gradient elution

Table 3 : The retention time of the components that modify HPLC mode (70:30)
ratio of (ACN : Water )
Discussion
High-performance liquid chromatography or high-pressure liquid
chromatography (HPLC) is a chromatographic method that is used to separate a
mixture of compounds in analytical chemistry and biochemistry so as to identify,
quantify or purify the individual components of the mixture. Reversed phase HPLC
(RP-HPLC) has a non-polar stationary phase and an aqueous, moderately polar
mobile phase.

The composition of the mobile phase is either maintained as a constant or as

varied during the chromatographic analysis. The constant approach is effective for the
separation of the sample components that are not very dissimilar in their affinity for
the stationary phase. If the polarity of the stationary phase and compound are similar,
the retention time increases because the compound interacts stronger with
the stationary phase.

Polar compounds have long retention times on polar stationary phases and
shorter retention times on non-polar columns using the same temperature. The least of
interaction with the stationary phase or the most amount of interaction with the
mobile phase will exit the column faster. 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.

In this experiment, is to optimize the separation of mixture of 5

compounds(caffeine, acetone, methyl benzoate, phenatole, phenantrene) using HPLC
method development by varying the mobile phase composition. For the mobile phase,
a mixture of acetonitrile:water is used. Both isocratic elution and gradient elution are
used in this experiment. Isocratic elution were first done for mobile phase
composition of ACN:H2O 70:30 to identify the components in the mixture. Then,
gradient elution was done to improve the efficiency of the column so that the later
eluting compounds will elute faster.
 The first step of the experiment is to find the suitable mobile phase composition.
A mobile phase composition of ACN:H2O of 50:50 and 70:30 were done. The
resolution for both mobile phase compositions of the standard mixture is above 1.5,
50:50 mobile phase compositions has a resolution of 7.0156 and 70:30 mobile phase
composition has a resolution of 6.1078 which are an ideal separation or complete
separation between peaks.

But the elution time for the mobile phase composition of 50:50 has a longer
elution time for the last or late eluting compound, at the 26 minutes but the elution
time of last eluting compound for the mobile phase composition of 70:30 ACN:H20 is
at the 6 minutes, so this mobile phase ratio is chosen since it is more suitable. In other
words, the mobile phase composition of 70:30 ACN:H20 has shorter analysis time.

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 using
the selected HPLC conditions (ACN:H2O 70:30). The composition of the mobile
phase remains constant throughout the HPLC separation, the separation is deemed an
isocratic elution. By this technique, we know and found out that caffiene eluted at the
1.121 minutes, acetone at the 1.332 minutes, methyl benzoate at the 2.095 minutes,
phenatole at the 2.812 minutes and phenantrene at the 6.353 as shown in the
chromatogram of the individual injection of the compound. The mobile phase is polar
and the stationary phase is non-polar, it will retain non-polar compounds and less
retain polar compounds. By that, the most polar compound is caffiene, followed by
acetone, methyl benzoate, phenatole and the least polar and non-polar compound is
phenantrene.

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. Based on the result, the composition of
mobile phase, acetonitrile:water changed from 50:50 to 70:30 then followed by 85:15
has a resolution which is 5.7411. Thus it gives a shorter time for the elution and well
separation between peaks.

Conclusion
The optimized mobile phase composition for the separation of the mixture is 70:30
(ACN: H2O) by using isocratic elution hence gives resolution of 6.1078. Thus well
separation between peaks obtained. Higher composition of organic solvent will
increase the solvent strength that will shorten the analysis time. In gradient elution,
the composition of mobile phase, acetonitrile:water changed from 50:50 to 70:30
then followed by 85:15 has a resolution which is 5.7411 which gives good resolution,
better separation, and high efficiency in separating the mixture. The most polar
compound is caffiene, followed by acetone, methyl benzoate, phenatole and the least
polar and non-polar compound is phenantrene.

References
1. Nor’ashikin Saim, Ruziyati Tajuddin., Mardiana Saaid, Rozita Osman, (2019),
Analytical Separation Methods Laboratory Guide (3nd edition).
2. G.P. Thomas, (2019), High Performance Liquid Chromatography (HPLC) –
Methods, Benefits and Applications, Article of Azo Material.
3. Retrieved from : Journal and books of Essentials in Modern HPLC (2013)
https://www.sciencedirect.com/topics/chemistry/reverse-phase-liquid-c
hromatography