CHM 510 Experiment 2
CHM 510 Experiment 2
CHM 510 Experiment 2
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
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
Reverse
Phase
HPLC
the
stationary
phase
is
nonpolar
EXPERIMENTAL PROCEDURE
a.
b.
c.
d.
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
2.
B.
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
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
0.973
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
2.
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
3.
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
Resolutio
n of 2
peaks
(peak 2
& peak
3)
18
4.
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
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,
HPLC
method
development
by
varying
the
mobile
phase
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