EXPERIMENT 2: HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) :

METHOD DEVELOPMENT

NAME

MUHAMMAD RAHIMI BIN ZAHURI

(2015829866)

PARTNERS NAME

AKMAL ARSYAD BIN MOHD RAFFI

(2015875404)

MOHD IQBAL BIN NORAZMAN

(2015840192)

MUHAMMAD RAHIMI BIN ZAHURI

(2015829866)

SYARAFUDDIN BIN MOHD SHAMSIBY

(2015872898)

DATE OF EXPERIMENT:
DATE OF SUBMISSION OF REPORT:

Introduction:
There are three objectives of this experiment. Firstly, the objective is to study the development
for optimizing the separation of mixture of five compounds which are standard mixtures of
caffeine, acetone, methyl benzoate, phenatole, and phenanthrene using HPLC by varying the
mobile phase composition. Second, to identify the individual components exist in the mixture
and third is to optimize the separation between each peaks using gradient elution method. HPLC
is the most widely used of all of the analytical separation technique. This type of instrument is
suitable for the separation of nonvolatile compounds. For this experiment, reverse-phase is used
at which stationary phase is non polar while the mobile phase is polar. This will make the most
polar component to be eluted first. In the liquid chromatography, it is more complicated due to
the interaction of the components of analyte with both the stationary phase and the mobile phase.
By that, increasing the mobile phase polarity will increases the retention time of the components.
The polarities of various analytes functional groups in increasing order are as shown below:
Hydrocarbon < Ether < Ester < Ketones < Aldehydes < Amides < Amines < Alcohols
Water is the most polar compounds among the compounds containing any of the preceding
functional groups. In this experiment, 5 compounds which are caffeine, acetone, methyl
benzoate, phenatole, and phenanthrene are determined by varying the mobile phase composition
throughout the analysis. This is known as gradient elution technique. Gradient elution is a
condition at which the composition of the mobile phase is changed during the separation. Two or
more solvents that differ in polarity are employed. The method was intended to decrease the
retention time for the later eluting components so that they can elute faster which then produces
narrower peak thus improves the peak shape. This experiment uses water/acetonitrile gradient at
which more hydrophobic components will elute when the mobile phase consists of mostly
acetonitrile which gives a relatively hydrophobic mobile phase. The more hydrophilic
compounds will elute under conditions of relatively low acetonitrile and high water.

Experimental:
Instrument used: Liquid chromatograph (Agilent G1314A HPLC) equipped diode array detector
(DAD) and 5m RP C18 column and 20L sample loop.
a. Instrument set up
Detector wavelength : 254nm
Flow rate
: 1.5mL min-1
Mobile phase
: acetonitrile : water
b. Effect of mobile phase on HPLC separation.
The instrument was set up with the mobile phase ratio acetonitrile:water (50:50 v:v) and
the sample was injected into the column. The mobile phase composition was then
changed to acetonitrile:water (70:30 v:v) and the sample was injected into the column.
The resolution for both the composition was calculated and compared. The suitable
composition of mobile phase of these compounds is identified.
c. Identification of components in the mixture.
Each individual compound was injected into the column and the component of the mixture
was identified using the selected HPLC conditions.
d. Separation using gradient elution.
Based on the separation above, the gradient elution method is performed in order to
improve the column efficiency. The suitable ratio of the mobile phase is set up and the
sample was injected into the column. The method was repeated until a suitable ratio of
mobile phase was identified and all of the peaks were separated well with short analysis of
time.

Results and Discussion:

Resolution (Isocratic Elution)

Mobile phase composition: ACN:H2O (50:50 v:v)
Peak

Calculation

Resolution, Rs

1-2

2( 1.1260.978)
=
0.0551+0.0681

2.40

2-3

2( 3.2511.126)
=
0.0551+0.1068

26.30

3-4

2( 5.5523.251)
=
0.1548+0.1068

17.59

4-5

2( 20.5855.552)
=
0.5191+0.1548

44.61

Table 1.1 Resolution Run 1

Mobile phase composition: ACN:H2O (70:30 v:v)

Peak

Calculation

Resolution, Rs

1-2

2( 1.0570.957)
=
0.0591+0.0520

1.80

2-3

2( 1.7361.057)
=
0.0650+0.0520

11.61

3-4

2(2.3111.736)
=
0.0650+ 0.0837

7.73

4-5

2( 5.1732.311)
=
0.2342+ 0.0837

18.00

Table 2.1 Resolution Run 1

Peak

Calculation

Resolution, Rs

1-2

2( 1.0590.959)
=
0.0496+0.0558

1.90

2-3

2( 1.7401.059)
=
0.0600+0.0496

12.43

3-4

2( 2.3151.740)
=
0.0753+0.0600

8.50

4-5

2(5.1662.315)
=
0.2192+0.0600

20.42

Table 2.2 Resolution Run 2

Component

Retention Time

Width

Caffeine

0.960

0.0614

Acetone

1.059

0.0513

Methyl Benzoate

1.739

0.0652

Phenatole

2.315

0.0779

Phenanthrene

5.175

0.1307

Table 3.0 Retention time for individual component

Resolution (Gradient Elution)

Peak

Calculation

Resolution, Rs

1-2

2( 1.1280.981)
=
0.0714+0.0540

2.34

2-3

2( 2.4171.128)
=
0.0531+0.0540

24.07

3-4

2( 2.7992.417)
=
0.0550+0.0531

7.07

4-5

2( 4.0162.799)
=
0.1448+ 0.0550

12.18

Table 5.0 Resolution for Gradient Elution

Discussion:

In the experiment, a High Liquid Performance Chromatography (HPLC) Agilent G1314A was
used. The detector used was diode array detector (DAD) which included 5m RP C 18 column and
20L sample loop. HPLC is a method or technique to separate, identify, and quantify the
components in a mixture. It is suitable for nonvolatile compounds, thermally unstable, and has
high molecular weights. At the flow rate of 1.5mL min -1 and the detector of 254nm, the mobile
ratio was set up at 50% acetonitrile and 50% water at the beginning of the experiment in order to
analyze the effect of mobile phase composition on the chromatography separation. After the
standard mixture is injected, the process is run and the peak obtained is analyzed. Then the ratio
was changed to 70:30 respectively on the same mobile phase. The resolution of the
chromatogram is calculated by using the formula stated below:

R=

2(t 2t 1)
W 1+W 2

Where t is the retention time and W is the peak width.

From the experiment, we need to identify the components contained in the standard mixture by
using the optimized liquid chromatography conditions getting from the ratio of the mobile phase.
Other than that, gradient elution separation must also be conducted to improve the efficiency of
the column. This means that if the isocratic elution does not provide sufficiently rapid elution of
all the components, then gradient elution can be used in order to replace the method. From the
chromatogram obtained with the resolution calculated, it can be said that by comparing the
average resolution for both mobile phase compositions, we can see that the mobile phase ratio of
70% acetonitrile and 30% water gives the best resolution which is 1.80. This indicates that the
resolution is good and the efficiency of the separation increases. Meanwhile, for the mobile
phase ratio of 50% acetonitrile and 50% of water, the resolution is 2.40 which is also good
separation but less efficient due to the high analysis of time compared to the 70:30 ACN:water
ratio. This experiment also identified the components in the standard mixture when it is injected
individually by using the optimum mobile phase composition with the rati of 70:30 ACN:water.
This ratio is used due to the best composition baseline separation compared to the 50;50 ratio.
The compound in the standard mixture is identified by comparing the retention time of the
standard mixture with the retention time of the individual compound. From the chromatogram

obtained, it indicates that peak 1 is caffeine, followed by peak 2 which is acetone, peak 3 is
methyl benzoate, peak 4 is phenatole, and peak 5 is phenanthrene. From the chromatogram
obtained from both of the methods which are isocratic elution method and gradient elution
method, it can be compared that there are some differences from the condition of separation. In
isocratic elution, the peak width increases as the retention time increases. This leads to late
eluting peaks which is broad. Meanwhile by using gradient elution, the retention times will
decrease which will make the eluting components to elute faster, which produces narrower and
sharper peaks. This also reduces the tailing peak as the increasing concentration of the organic
eluent pushes the tailing part of a peak forward. Hence, this can be said that the gradient elution
method gave a shorter overall analysis with similar resolution of the peak compared to the
isocratic elution. From the results obtained, it shows that almost the entire peaks were well
separated but only the first peak and the second peak were not well separated. The first peak
indicates the caffeine compound while the second peak indicates the acetone compound. There
are some factors that lead to the problem to happen. In this case, one of the solution is by
degassing the mobile phase properly in order to remove the small air bubbles from getting inside
the tube. Such bubbles can cause noise in the detector response which then hinder the flow of the
mobile phase through the columns. For the problem to be solved, degassing of the mobile phase
must be done. Besides, when injecting the sample into the loop, the injection volume must not be
above the volume indicated on the loop. A flat end needle must also be used instead of the others
in order to prevent the damage of the injection port. Besides, the syringe must also be washed for
at least 5 times by using the washing solvent and must be rinsed with the standard to be injected
for at least 3 times in order to avoid any contaminants from entering the syringe which may
cause the undesired peak to appear in the chromatogram.

Conclusion:
From the experiment, it can be concluded that by varying the mobile phase composition in
HPLC, the method development of optimizing a separation of a standard compounds can be
determined.

Reference:

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Saim, N., Tajuddin, R., & Saaid, M. (2014). Experiment 1 Gas
Chromatography(GC): Optimization of Flow Rate and Column
Temperature. In ANALYTICAL SEPERATION METHODS LABORATORY
GUIDE 2nd edition (pp. 1-2). Selangor: UiTM Press.
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