BACHELOR OF SCIENCE (HONOR) APPLIED CHEMISTRY (AS245)

ANALYTICAL SEPARATION METHOD

CHM510
EXPERIMENT 2: High Performance Liquid Chromatography
(HPLC): Method Development

NAME NURANISAH NADIAH BINTI MOHD NIZAM

STUDENT ID 2022855638

GROUP MEMBERS
1) NUR AMIRAH BALQIS BINTI MOHD KOSIM
(2022800388)
2) AINUL SYARAFANA BINTI SHAMSUL HAYAT
(20224942820)
3) NURATHIRAH NABIHAH BINTI SAIRI (20224942820)
4) NUR HIDAYAH MOHD NAJIB (2022

CLASS AS2452S1

NAME OF LECTURER DR WAN NAIHAH WAN IBRAHIM

a. Introduction

High-performance liquid chromatography (also known as High pressure liquid

chromatography, or HPLC) is a particular type of column chromatography that is frequently
used in biochemistry and analysis to separate, identify, and quantify the active chemicals. The
primary components of HPLC are a column that retains packing material (stationary phase), a
pump that circulates one or more mobile phases through the column, and a detector that
displays the retention periods of the molecules. The interactions between the stationary phase,
the molecules being analysed, and the solvent(s) being utilised affect retention time. The
analysis sample is added in a small amount to the stream of mobile phase and is slowed down
by particular chemical or physical interactions with the stationary phase. The analyte's
characteristics as well as the composition of the stationary and mobile phases affect the
amount of retardation. The retention time is the amount of time it takes for a certain analyte
to elute, or leave the column. A miscible mixture of water or organic liquids is a frequent type
of solvent (the most common are methanol and acetonitrile). Gradient elution is the
separation process used to change the mobile phase's composition during the analysis.
Depending on how well the analyte binds to the present mobile phase, the gradient separates
the analyte mixtures. Depending on the characteristics of the stationary phase and the analyte,
the choice of solvents, additives, and gradient will differ.

Types of HPLC

a. Normal phase chromatography, also called Normal phase HPLC (NP-HPLC), is a

technique for polarity-based analyte separation. Polar stationary phase and non-polar mobile
phase are both used in NP-HPLC. The polar stationary phase reacted with the polar analyte
and held it. Increased analyte polarity results in stronger adsorption forces, and the interaction
of the polar analyte with the polar stationary phase lengthens the elution time.
b. Reversed-phase chromatography: Reversed-phase A non-polar stationary phase and an
aqueous, moderately polar mobile phase are the components of HPLC (RP-HPLC or RPC).
Using a polar eluent, a comparatively non-polar analyte, and a non-polar stationary phase,
repulsive forces are produced that lead to hydrophobic interactions, which are the basis of
RPC. Upon interaction with the ligand in the aqueous eluent, the analyte's affinity for the
stationary phase is proportional to the contact surface area around its non-polar segment.

c. Ion exchange chromatography: The attraction between solute ions and charged sites
attached to the stationary phase is what generates retention in this type of chromatography.
Same-charge ions are not included. This type of chromatography is frequently employed in
the ion-exchange chromatography of proteins, the high-pH anion-exchange chromatography
of carbohydrates and oligosaccharides, the ligand-exchange chromatography, the ion-
exchange chromatography of proteins, etc.

d. Bio-affinity chromatography: Proteins and ligands are separated based on a specific,

reversible interaction. A bio-affinity matrix has ligands covalently bonded to a solid support
that holds onto proteins that interact with the ligands attached to the column.

b. Objectives

To optimise a separation of 5 compounds using HPLC by verifying the mobile phase

composition.

c. Procedure

1. The instrument wavelength was set up to 254 nm and the flow rates set 1.5 mL min -1.
Acetonitrile and water are used as the mobile phase.

2. The instrument was set to use a mobile phase ratio: water (50: 50 v: v) and the sample
was injected.
3. Then, the mobile phase composition was changed to 70:30. The best composition of
mobile phase finally is chosen. Each component was injected individual to identify the
component mixture using the selected HPLC conditions.

4. Based on the separation, a gradient elution separation was performed to improve the
efficiency of the column

d. Data and results

Isocratic elution of ratio 70% H₂O: 30% ACN

No. of Peak Retention Width Resolution Average

injection time (min) (min) (Rs) resolution
(Rs)
1st 4 1.880 0.719
15.776
5 3.699 0.1587
17.899
2nd 4 1.879 0.0717
20.022
5 3.738 0.1140

Isocratic elution of ratio 50% H₂O: 50% ACN

No. of Peak Retention time Width (min) Resolution

injection (min) (Rs)
1st 3 4.316 0.1660
31.867
4 13.868 0.4335
The effect of the gradient elution

0 min (80:20)

1 min (90:10)

2.5 min (85:15)

No. of Peak Retention Width Resolution Average resolution

injection time (min) (min) (Rs) (Rs)
1st 4 1.747 0.0858
8.9675
5 2.520 0.0866

2nd 4 1.752 0.0858

8.9623 7.0382

5 2.525 0.0867

3rd 3 1.472 0.0869

3.1847
4 1.747 0.0858

The retention time for standard compound

Sample Retention time (min)

Caffeine 1.001
Acetone 1.048
Methyl benzoate 1.492
Phenetole 1.877
Phenanthrene 3.724
Sample calculation:
Sample calculation for resolution (Rs)
2(tR ₂−tR ₁)
Rs =
w ₂+ w ₁

2(3.699−1.880)
Rs {₄, ₅} [ Isocratic Elution for 1st Injection] =
0.0719+0.1587
for composition 70:30 H2O: ACN
= 15.776

15.776+20.022
Average Rs [ Isocratic Elution] = = = 17.899
2

e. Discussion

The column utilised in this experiment was C18. Mobile phase is made up of polar
solvents like water and acetonitrile. A nonpolar column, or hydrophobic column, is the
C18 column. According to the data, a reversed phase was used for the separation modes.
When the solvent strength is greater in reversed phase, the compound's polarity is reduced.
Therefore, less polar compounds will elute quicker than polar compounds.

By adjusting the mobile phase composition, it was possible to improve the separation of a
combination of five compounds—caffeine, acetone, methyl benzoate, phenatole, and
phenanthrene using HPLC. ACN: H 2O composition was used as a combination for the
mobile phase. In this experiment, gradient and isocratic elution are both used. To initially
isolate the components in the mixture, isocratic elution was used to analyse the
composition of the mobile phase. Gradient elution was then carried out to increase the
column's effectiveness and speed up the elution of chemicals that would otherwise take
longer.

At the start of the separation process, the ratio of ACN: H 2O in the mobile phase was fixed
at 50/50. To observe the separation of the compounds, the ratio was then altered to ACN:
H2O 70:30. If the mobile phase's composition was sufficient for compound separation, it
could be used to achieve a better separation. By comparing the retention times of the
standard mixture with the various standards, the compound was identified after the
standard mixture was injected into the column.

At composition of mobile phase (ACN: H2O) 50:50, the resolution for peak 3 and peak 4
is 31.867. However, the resolution is greater than 1.5 baseline resolution so it produces
broad peak. This resolution needs to be improved until it achieves to the 1.5. As we can
see, the mobile phase composition for (ACN: H2O) 50:50 has a longer elution time for the
last compound which reaches to 13.868 min.

After that, we change the composition H 2O: ACN mobile phase to 70:30. At this
composition, we can see from the result that the resolution for peak 4 and peak 5 besides
peak 4 and peak 5 are 15.776 and 20.022. The values are still greater than 1.5 baseline
resolution so it produces broad peaks. However, 70:30 composition ACN: H2O has a
shorter elution time compared to composition 50:50 which is 3.738 min for the last
compound to elute.

According to the data, a mobile phase ratio of (ACN: H2O) 70:30 provides better
separation than a ratio of 50:50, which does not effectively separate the combination. This
is due to the fact that the polarity of the mobile phase will have an impact on how quickly
the analyte or compound to be separated will elute. The elution duration of the non-polar
analyte will be shortened by the mobile phase's less polar characteristics because doing so
will enhance the solvent and eluent strength.

By gradually or continually modifying the composition of the mobile phase throughout the
analysis, compounds can be separated more efficiently using gradient elution. Moving the
mixture's highly retained components more quickly while keeping the least retained
component well resolved is the target. To separate the least retained component, begin
with a low solvent strength (ACN: H2O) 50:50 mobile phase. So that the strongly retained
compound elutes faster, gradually raise the solvent strength to ACN: H2O 70:30. This
elution method will result in a shorter retention period for the analytes to be eluted from
the column and more efficient separation.
f. Conclusion

The experiment's objective was determined, and a 70:30 H 2O: ACN mobile phase
composition was found to be ideal for separating the mixture. Solvent strength will rise
with increased organic solvent composition, reducing analytical time. Thus, caffeine is the
initial peak, which is followed by peaks for acetone, methyl benzoate, phenatole, and
phenanthrene.

g. Reference

P. Jandera, J. C. (1985).
Gradient Elution in Column
Liquid Chromatography:
Theory and
Practice. Journal of
Chromatography Library, 67-
69.
P. Jandera, J. C. (1985).
Gradient Elution in Column
Liquid Chromatography:
Theory and
Practice. Journal of
Chromatography Library, 67-
69.
 P. Jandera, J. C. (1985). Gradient Elution in Column Liquid Chromatography:
Theory and Practice. Journal of Chromatography Library, 67-69.
 INTRODUCTION to High Performance Liquid Chromatography (HPLC)
https://www.researchgate.net/publication/
303812584_INTRODUCTION_to_High_Performance