CHM 510

ANALYTICAL SEPARATION METHODS

EXPERIMENT 2
HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY (HPLC): METHOD
DEVELOPMENT
NAME: NABILAH BINTI ABD RAHMAN
STUDENT ID: 2015484718
LAB PARTNERS: 1. ANIZA BINTI ABDULLAH (2015827038)
2. NIK NURFARAHAIN RIFHAN BINTI NIK AZMAN
(2015896328)
GROUP: AS2453D1
LECTERURS NAME: DR. MARDIANA BINTI SAAID
DATE PERFORMED: 30TH MARCH 2016
DATE OF SUBMISSION: 27TH MAY 2016

TITLE OF EXPERIMENT
Experiment 2 High Performance Liquid Chromatography (HPLC): Method
Development

OBJECTIVE
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.

INTRODUCTION
Chromatography is a technique to separate mixtures of substances
into their components on the basis of their molecular structure and
molecular composition. This involves a stationary phase (a solid, or a
liquid supported on a solid) and a mobile phase (a liquid or a gas). The
mobile phase flows through the stationary phase and carries the
components of the mixture with it. Sample components that display
stronger interactions with the stationary phase will move more slowly
through the column than components with weaker interactions. This
difference in rates causes the separation of various components.
Chromatographic separations can be carried out using a variety of
stationary phases, including immobilized silica on glass plates (thin-layer
chromatography), volatile gases (gas chromatography), paper (paper
chromatography) and liquids (liquid chromatography).
High performance liquid chromatography (HPLC) is basically a highly
improved form of column liquid 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. All

chromatographic separations, including HPLC operate under the same basic

principle; separation of a sample into its constituent parts because of the
difference in the relative affinities of different molecules for the mobile phase
and the stationary phase used in the separation.

Normal Phase HPLC method separates analytes on the basis of

polarity. NP-HPLC uses polar stationary phase and non-polar mobile phase.
Therefore, the stationary phase is usually silica and typical mobile phases
are hexane, methylene chloride, chloroform, diethyl ether, and mixtures of
these. Polar samples are thus retained on the polar surface of the column
packing longer than less polar materials.
In

Reverse

Phase

HPLC

the

stationary

phase

is

nonpolar

(hydrophobic) in nature, while the mobile phase is a polar liquid, such as

mixtures of water and methanol or acetonitrile. It works on the principle of
hydrophobic interactions hence the more nonpolar the material is, the
longer it will be retained.
In Size-exclusion HPLC the column is filled with material having
precisely controlled pore sizes, and the particles are separated according
to its their molecular size. Larger molecules are rapidly washed through
the column; smaller molecules penetrate inside the porous of the packing
particles and elute later.
Ion-Exchange HPLCs stationary phase has an ionically charged
surface of opposite charge to the sample ions. This technique is used
almost exclusively with ionic or ionizable samples. The stronger the
charge on the sample, the stronger it will be attracted to the ionic surface
and thus, the longer it will take to elute. The mobile phase is an aqueous
buffer, where both pH and ionic strength are used to control elution time.

EXPERIMENTAL PROCEDURE
a.

HPLC was set up with following condition:

Detector wavelength: 254nm
Mobile phase flow rate: 1.5mL/min
Mobile phase: acetonitrile:water

b.

Effect of mobile phase on HPLC separation:

The standard mixture was injected into the HPLC by using mobile
phase composition of acetonitrile:water (50:50 v:v). The mobile
phase ratio of acetonitrile:water was then changed for the second
injection to 70:30. The resolution of the two chromatograms were
calculated and compared to determine the best composition for that
analysis.

c.

Identification of components in the mixture:

Each of the standard compounds (caffeine, methyl benzoate,
phenatole and phenantrene) was injected individually with the
optimized HPLC conditions to be compared with the retention time
of the standard mixture.

d.

Separation using gradient elution:

Gradient elution separation was performed based on the separation
by using isocratic elution to improve the efficiency of the column.

EXPERIMENTAL RESULTS, DATA AND CALCULATIONS

A.

Effect Of Mobile Phase On HPLC Separation

Isocratic Elution of Standard Mixture

Mobile
Phase
Ratio
(Compositi
on) of
ACN:water

50:50

70:30

Peaks

Retentio
n Time of
Peaks
(min)

Base
Peak
Width of
Peaks
(min)

Area of
Peaks
(mAU*s)

Peak 1

0.975

0.0637

Peak 2

1.118

0.0593

Peak 3

3.253

0.1115

Peak 4

5.582

0.1563

Peak 5

20.672

0.5183

1.17225e
4
484.1832
9
5215.045
41
4499.707
03
1.30105e
5

Peak 1

0.956

0.0528

8957.484
9

Resolutio
n of 2
peaks
(peak 2
& peak
3)

25

(Average
from
injection 1
and injection
2)

Peak 2

1.056

0.0503

Peak 3
Peak 4

1.728
2.296

0.0602
0.0716

Peak 5

5.053

0.2088

1708.031
56
4607.312
4266.629
15
4.51238e
4

12.16

Calculation of resolution of 2 peaks:

1.

Mobile Phase Ratio (Composition) of ACN:water ; (50:50)

Rs(2,3) = 2( Rt,3- Rt,2 )
(Wb,2 + Wb,3)
Rs(2,3) = 2( 3.253 - 1.118 )
(0.0593 + 0.1115)
= 25 #

2.

Mobile Phase Ratio (Composition) of ACN:water ; (70:30)

Rs(2,3) = 2( Rt,3- Rt,2 )
(Wb,2 + Wb,3)
Rs(2,3) = 2( 1.728 - 1.056 )
(0.0503 + 0.0602)
= 12.16 #
The resolution for both mobile phase compositions of the standard

mixture is above 1.5, which is and 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, the

mobile phase composition of 70:30 ACN:H 20 is chosen. In other words, the

mobile phase composition of 70:30 ACN:H20 has shorter analysis time.

B.

Identification Of Each Component In The Mixture

Mobile phase composition of ACN:water is 70:30

1.

Caffeine

2nd
Injection
2.

Retention
time in
standard
(min)
1.056

Retention
time in
Mixture
(min)
1.057

Retention
time in
standard
(min)
1.728

Retention
time in
Mixture
(min)
1.729

Retention
time in
standard
(min)
2.296

Retention
time in
Mixture
(min)
2.270

Area
(mAU*s)
8916.14160

Area
(mAU*s)
501.43103

Methyl benzoate

1st Injection
4.

Retention
time in
Mixture
(min)
0.962

Acetone

2nd
Injection
3.

Retention
time in
standard
(min)
0.956

Area
(mAU*s)
4969.65430

Phenetole

1st Injection

Area
(mAU*s)
1912.42883

5.

Phenantrene

1st Injection

C.

Retention
time in
standard
(min)
5.053

Retention
time in
Mixture
(min)
4.227

Area
(mAU*s)
3546.25146

Separation Using Gradient Elution

Gradient Elution of Standard Mixture

1.

1st injection of standard mixture for gradient elution

0 min : (70:30)
0 2 min : (75:25)
2 3 min : (80:20)

3 5 min : (80:20)
Mobile
Phase
Ratio
(Compositi
on) of
ACN:water

50:50

Peaks

Retentio
n Time of
Peaks
(min)

Base
Peak
Width of
Peaks
(min)

Area of
Peaks
(mAU*s)

Peak 1

0.973

0.0540

Peak 2

1.120

0.0529

Peak 3

2.144

0.0513

Peak 4

2.798

0.0627

Peak 5

4.992

0.1496

8783.035
16
1196.049
93
3055.491
46
3080.991
21
2.74360e
4

Calculation of resolution of 2 peaks

Rs(2,3) = 2( Rt,3- Rt,2 )
(Wb,2 + Wb,3)
Rs(2,3) = 2( 2.144 - 1.120 )
(0.0529 + 0.0513)
= 20 #

2.

2nd injection of standard mixture for gradient elution

0 min : (70:30)
0 2 min : (70:30)
2 3 min : (80:20)

Resolutio
n of 2
peaks
(peak 2
& peak
3)

20

3 4 min : (80:20)
Mobile
Phase
Ratio
(Compositi
on) of
ACN:water

50:50

Peaks

Retentio
n Time of
Peaks
(min)

Base
Peak
Width of
Peaks
(min)

Area of
Peaks
(mAU*s)

Peak 1

0.977

0.0622

Peak 2

1.123

0.0548

Peak 3

2.117

0.0552

Peak 4

2.695

0.0632

Peak 5

4.710

0.1408

1.11516e
4
1712.522
58
4358.705
57
4370.255
86
3.07807e
4

Calculation of resolution of 2 peaks

Rs(2,3) = 2( Rt,3- Rt,2 )
(Wb,2 + Wb,3)
Rs(2,3) = 2( 2.117 - 1.123 )
(0.0548 + 0.0552)
= 18 #

3.

3rd injection of standard mixture for gradient elution

Resolutio
n of 2
peaks
(peak 2
& peak
3)

18

0 min : (70:30)
0 1.5 min : (80:20)
1.5 4 min : (80:20)
Mobile
Phase
Ratio
(Compositi
on) of
ACN:water

50:50

Peaks

Retentio
n Time of
Peaks
(min)

Base
Peak
Width of
Peaks
(min)

Area of
Peaks
(mAU*s)

Peak 1

0.970

0.0658

Peak 2

1.116

0.0550

Peak 3

2.070

0.0533

Peak 4

2.553

0.0588

Peak 5

4.220

0.1414

1.16349e
4
1840.950
56
4705.826
17
4711.999
02
3.04860e
4

Calculation of resolution of 2 peaks

Rs(2,3) = 2( Rt,3- Rt,2 )
(Wb,2 + Wb,3)
Rs(2,3) = 2( 2.070 - 1.116 )
(0.0533 + 0.0550)
= 18 #

Resolutio
n of 2
peaks
(peak 2
& peak
3)

18

4.

4th injection of standard mixture for gradient elution

0 min : (70:30)
0 1 min : (80:20)
1 3.5 min : (80:20)

Mobile
Phase
Ratio
(Compositi
on) of
ACN:water

50:50

Peaks

Retentio
n Time of
Peaks
(min)

Base
Peak
Width of
Peaks
(min)

Area of
Peaks
(mAU*s)

Peak 1

0.970

0.0562

Peak 2

1.116

0.0514

Peak 3

2.037

0.0500

Peak 4

2.469

0.0529

Peak 5

4.116

0.1522

9628.161
13
1393.817
38
3498.078
61
3560.180
18
2.80843e
4

Calculation of resolution of 2 peaks

Rs(2,3) = 2( Rt,3- Rt,2 )
(Wb,2 + Wb,3)
Rs(2,3) = 2( 2.037 - 1.116 )
(0.0500 + 0.0514)
= 18 #

Resolutio
n of 2
peaks
(peak 2
& peak
3)

18

DISCUSSION
High Performance Liquid Chromatography (HPLC) is a form of
column chromatography that pumps a sample mixture or analyte in a
solvent (known as the mobile phase) at high pressure through a column
with chromatographic packing material (stationary phase). A moving
carrier gas stream of helium or nitrogen carries the sample. HPLC has the
ability to separate, and identify compounds that are present in any sample
that can be dissolved in a liquid in trace concentrations as low as parts
per trillion. Because of this versatility, HPLC is used in a variety of
industrial

and

scientific

applications,

such

as

pharmaceutical,

environmental, forensics, and chemicals.

Sample retention time will vary depending on the interaction
between the stationary phase, the molecules being analyzed, and the
solvent, or solvents used. As the sample passes through the column it
interacts between the two phases at different rate, primarily due to
different polarities in the analytes. 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 faster.
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:H 2O
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:H 2O 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 25 and 70:30 mobile phase composition has a resolution of
12 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 20 th
minute but the elution time of last eluting compound for the mobile phase
composition of 70:30 ACN:H20 is at the 5th minute, 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 second step is to inject the compounds individually (caffeine,
acetone, methyl benzoate, phenatole, phenantrene) to identify the
components in the mixture using the selected HPLC conditions (CAN:H 2O
70:30). If 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 caffeine eluted at
the 0.9th minute, as shown in the chromatogram of the individual injection
of the compound. Then, the 2nd eluting compound is acetone at 1st minute,
also shown in the chromatogram of the chromatogram of the individual
injection of the compound. The 3 rd eluting compound would be methyl
benzoate, at 1.7th minute, shown at the chromatogram of the individual
injection of the compound. Phenetole is the 4th eluting compound, at the
2nd minute, as shown in the chromatogram of the individual injection of
the compound and the last and 5 th eluting compound is phenantrene, at
5th minute, as shown in the chromatogram of the individual injection of the

compound. Since 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 caffeine, followed by
acetone, methyl benzoate, phenatole and the least polar or non-polar
compound is phanantrene.
Often the only way to elute all of the compounds in the sample in a
reasonable amount of time, while still maintaining peak resolution, is to
change the ratio of polar to non-polar compounds in the mobile phase
during the sample run. Known as gradient chromatography, this is the
technique of choice when a sample contains components of a wide range
of polarities. This is the third and last step in this experiment. For a
reverse phase gradient, the solvent starts out relatively polar and slowly
becomes more non-polar. The gradient elution offers the most complete
separation of the peaks, without taking an inordinate amount of time. A
sample containing compounds of a wide range of polarities can be
separated by a gradient elution in a shorter time period without a loss of
resolution in the earlier peaks or excessive broadening of later peaks.
However, gradient elution requires more complex and expensive
equipment and it is more difficult to maintain a constant flow rate while
there are constant changes in mobile phase composition. Gradient elution,
especially at high speeds, brings out the limitations of lower quality
experimental apparatus, making the results obtained less reproducible in
equipment already prone to variation. If the flow rate or mobile phase
composition fluctuates, the results will not be reproducible. In this
experiment, gradient elution was done to improve the efficiency of the
column. This is done so that the late eluting compounds will elute faster,
and the results shown in the results and data section showed that the 4 th
injection for gradient elution is most suitable and the best. Since the
response factor is above 1.5 (18) it is still deemed an efficient and
complete separation. So, the gradient elution for the 4 th injection is set at
0 min (70:30), 0 1 min (80:20) and 1 3.5 min (80:20).

Conclusion
The most polar compound is caffeine (0.9 th minute), followed by acetone
(1st minute), methyl benzoate (1.7th minute), phenatole (2nd minute) and
the least polar or non-polar compound is phanantrene (5 th minute) and a
mobile phase composition of ACN:H2O is 70:30.
References
1.

http://laboratoryinfo.com/hplc/

2.

http://www.chemguide.co.uk/analysis/chromatography/hplc.html