UNIVERSITY TECHNOLOGI MARA CAWANGAN KUALA PILAH, NEGERI

SEMBILAN

ANALYTICAL SEPARATION METHODS (CHM510)

LAB REPORT
EXPERIMENT 2: HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC):
METHOD DEVELOPMENT

PREPARED BY:

NAMA STUDENT ID

EZAT NASHRUL BIN LATIF 2023687094

NUR ILYA NATASYA BINTI MOHD SABRI 2023479156

QASDINA AMIRA BINTI ABD RASHID 2022818794

JULIANI BINTI ALIAS 2022882748

PREPARED FOR:
DR. NUR NADIA BINTI DZULKIFLI

DATE OF SUBMISSION:
24 MAY 2024
OBJECTIVE:

1. To enhance the effect of mobile phase composition on HPLC separation using

isocratic elution.
2. To separate compounds using gradient elution to improve the efficiency of the
separation.

INTRODUCTION:

A comprehensive analytical chemistry method called high-performance liquid

chromatography (HPLC) is used to separate components in a chemical mixture. These
separations make use of a mobile phase that is forced by pressure to pass through a stationary
phase-packed column. A liquid sample is carried by the mobile phase through the column and
into the detector, where chemicals or analytes segregate as a result of differing interactions
with the stationary phase. After the analytes are eluted from the column, a detector monitors
them, and the observed signal is translated by a chromatography data system (CDS). A
chromatogram is the translated data output of HPLC analysis, where the y-axis measures a
particular signal produced by the detector and the x-axis represents time. It is possible to alter
the mobile phases using gradient elution or isocratic elution.

The mobile phase's composition is maintained constant during the elution process in an
isocratic elution. Gradient elution involves increasing the mobile phase's composition step-by-
step as the elution process progresses.

In HPLC, resolution is crucial to determine how quickly and how well-separated the
peak of the sample as they pass through the column. To determine the resolution, the difference
in retention time of peak is divided by the sum of peak width. The equation use to calculate the
resolution as below:

RS = 2 (tR2 – tR1) / W1 + W2

The compounds that were used in this experiment are caffeine, acetone, methyl
benzoate, phenatole and phenanthrene. Agilent Technologies HPLC was used in this
experiment to enhance the effect of mobile phase composition on HPLC separation using
isocratic elution and to separate compounds using gradient elution to improve the efficiency of
the separation.
REAGENTS AND SOLUTIONS:

HPLC grade acetonitrile

Deionised water
Standard mixture of caffeine, acetone, ethyl benzoate, phenatole and phenanthrene (around
100 ppm)

INSTRUMENT:

Liquid chromatograph (Agilent G1314A HPLC) equipped with diode array detector (DAD),
RPC18,a column and 𝜇L sample loop.

ANALYTICAL PROCEDURE:

1. The instrument set up as below:

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

2. Effect of mobile phase on HPLC separation.

The mobile phase using polar solvent which acetonitrile and deionized water with the
ratio 50:50. The standard mixture was injected. Then, the composition of mobile
phase was changed to ratio 70:30.

3. Identification of components in the mixture

To investigate the component in the mixture using the selected high performance
liquid chromatography or HPLC specification, each compound was injected
individually in automatic injection.

4. Separation using gradient elution

Based on the separation, gradient elution separation was performed to improve the
efficiency of the column by enhance the ratio of the column.
RESULT:

i. Effect of mobile phase on HPLC separation

In the experiment, we used two different ratios of mobile phase of acetonitrile : water to observe
the effect of mobile phase on HPLC separation. Initially, the ratio of 50:50 of ACN: H 2O was
used to separate the standard mixture solution. Then, the ratio of 70:30 of ACN: H 2O was used
to separate the standard mixture solutions. The retention time and resolution of the peak are
shown below.

Figure 1: Peak of the standard mixture solutions separated with 50:50 ACN:H 2O ratio
 Concentration Ratio Peak Retention Time (min) Peak Width (min)
of Mobile Phase
(ACN:H2O)

50:50 1 1.148 0.1100

2 2.663 0.1766

3 3.288 0.3511

4 4.312 0.2171

5 4.625 0.1754

6 5.405 0.5790

Table 1.0: Retention Rate (tR) of Peak of the standard mixture solutions separated with
50:50 ACN:H2O ratio

Concentration Ratio of Pairs of Adjacent Peak Resolution, Rs

Mobile Phase (ACN:H2O)

50:50 1&2 10.5722

2&3 2.3688

3&4 3.6044

4&5 1.594

5&6 2.0679

Table 1.1: Resolution (Rs) of Peak of the standard mixture solutions separated with 50:50
ACN:H2O ratio
Figure 2: Peak of the standard mixture solutions separated with 70:30 ACN:H 2O ratio

Concentration Ratio Peak Retention Time (min) Peak Width (min)

of Mobile Phase
(ACN:H2O)

70:30 1 1.102 0.1154

2 1.704 0.1563

3 2.077 0.1068

4 2.193 0.1289

5 4.122 0.1935

6 4.417 0.2252

Table 2.0: Retention Rate (tR) of Peak of the standard mixture solutions separated with
70:30 ACN:H2O ratio
 Concentration Ratio of Pairs of Adjacent Peak Resolution, Rs
Mobile Phase (ACN:H2O)

70:30 1&2 4.4313

2&3 2.8354

3&4 0.9843

4&5 11.9665

5&6 1.4091

Table 2.1: Resolution (Rs) of Peak of the standard mixture solutions separated with 70:30
ACN:H2O ratio
ii. Identification of components in the mixture

Individual compounds were injected in the HPLC machine by using the 70:30
ACN:H2O ratio as the mobile phase composition

Figure 3: Peak of the caffeine compound with 70:30 ACN:H2O ratio of mobile phase
composition

Figure 4: Peak of the acetone compound with 70:30 ACN:H2O ratio of mobile phase
composition
Figure 5: Peak of the methyl benzoate compound with 70:30 ACN:H2O ratio of mobile
phase composition

Figure 6: Peak of the phenatole compound with 70:30 ACN:H2O ratio of mobile phase
composition
Figure 7: Peak of the phenanthrene compound with 70:30 ACN:H2O ratio of mobile
phase composition

Compound Retention time (min) Peak Width (min)

Caffeine 1.071 0.0821

Acetone 1.142 0.0706

Methyl Benzoate 1.679 0.2306

Phenatole 1.626 0.0890

Phenantherene 4.2685 0.1945

Table 3: Retention time and Peak width of each individual compounds
DISCUSSION:

In this experiment, we used an isocratic elution in which the composition of the mobile phase
remains constant during the separation. Two solvents with different polarities, acetonitrile and
water, have been used as the mobile phase. Two compositions of mobile phase have been used
which are 50:50 and 70:30 (Acetonitrile: Water). Acetonitrile is an organic solvent that acts as
a modifier. Adding a surface modification to the eluent improves the interfacial compatibility
between the stationary and mobile phases and speeds up the analyte equilibration process
(Borai et al, 2008). The stationary phase was a non-polar column in which the c18 column was
attached to the silica surface. In Part A, the analyte used was an unknown mix while in Part B,
the analyte used was caffeine, acetone, methyl benzoate, phenetole and phenanthrene. Based
on the known polarities of the stationary phase and the mobile phase, we can determine that it
was in the reverse phase of partition chromatography HPLC. The instrument has been set up
using a detector wavelength 254 nm and the flow rate was 1.5µL min-1.

In Part A, which is the effect of the mobile phase on HPLC separations, the results
showed that the separation of the mixture using a 70:30 mobile phase composition is better and
more efficient than a 50:50 mobile phase composition. This is because, when we see the
chromatogram in Figure 1 and Figure 2, there are too many overlaps occurring if using a 50:50
composition rather than 70:30. By focusing on the third peak of the chromatogram for 50:50
and the second peak for 70:30, we can differentiate the efficiency of the separation. The reason
for choosing this peak is because it has a higher height of peak (a narrower peak), less overlap,
and has a good peak width. Based on Table 1.0 which uses a 50:50 composition, the retention
time for peak 3 is 3.288 min while in Table 2.0 which uses a 70:30 composition, the retention
time for peak 2 is 1.704 min.

Longer retention periods are the result of increasing the mobile phase's polarity. Lower
polarity mobile phases are necessary for shorter retention periods (Chemistry LibreTexts,
2021). Basically, the mobile phase for 70:30 is less polar than 50:50. Thus, we can see that
when the low polarity of the mobile phase is used, the solvent and eluent strength increase.
This causes the attraction between mobile phases and analytes to become stronger. Therefore,
the retention time or analysis time is decreased, which reduces the time of the analyte to elute
to a detector. Moreover, how well two elution peaks may be distinguished from one another in
a chromatographic separation is measured quantitatively by an elution's resolution. It is
calculated by dividing the total widths of the elution peaks by the difference in retention periods
between the two peaks. It is typically possible to properly separate the peaks if the resolution
is larger than unity. The resolution of the peaks using 50:50 composition mobile phase is 2.3688
while the resolution of the peaks using 70:30 composition mobile phase is 2.8354. Even though
both of these resolutions were more than 1.5, the peaks were not separated completely.
However, the peak at 70:30 mobile phase composition was near to being completely separated
than 50:50.

Next, in Part B, we have identified the components in the mixture of individual

compounds which are caffeine, acetone, methyl benzoate, phenetole, and phenanthrene using
the same composition of mobile phase which is 70:30. More polar solutes elute first in reversed-
phase HPLC, where the elution order is the opposite of that of normal-phase separation
(Chemistry LibreTexts, 2021). Based on the results obtained on Table 3, we can see that the
lowest retention time is 1.071 min which is for caffeine and the highest retention time is 4.2685
which is for phenanthrene. The reason for the difference in the retention time is because of the
polarity of the analytes. The polarity can be determined by the functional group and number of
carbons in the compounds. Based on Table 4, we can see that phenanthrene contained a greater
number of carbon atom in its structure making it to be highly non-polar. An interaction between
a highly non-polar analyte (phenanthrene) and the non-polar stationary phase (c18 column) is
very strong. The analytes are stronger remain in the stationary phase. Thus, the retention time
increase causes the analyte to take a longer time to elute to a detector.

Besides, the compound that eluted first was caffeine. Even though caffeine has 8
carbons in its structure, caffeine is a polar compound because it contains many nitrogen and
oxygen atoms. Thus, caffeine is classified as a highly polar compound. Therefore, the
interaction between highly polar analytes and the non-polar stationary phase is weaker. The
analytes will elute faster and have less retention time. For acetone, methyl benzoate, and
phenols, the acetone will elute first, followed by phenetole and methyl benzoate. Acetone
contains a carbonyl group, which causes it to be polar molecules but still less polar than
caffeine. Besides, phenetole and methyl benzoate both have the same number of carbon.
However, phenetole is more polar than methyl benzoate. Thus, phenetole is eluted first rather
than methyl benzoate.
 In addition, some errors occurred during this experiment since most of the peaks were
not separated well and many overlaps formed. There was a probability that peak contaminants
occurred during the experiment of mixed solution. There is a source of contamination
responsible for this. It is likely that the blank is contaminated if the contaminant peak seems to
rise or fall with varying blank injection volumes. If not, the system's other components cause
the pollution. Besides, this may be due to the highly absorbed and high interaction of mobile
phase.

Instead of using isocratic elution, this experiment can be improved using gradient
elution. Throughout the separation process, the mobile phase's elution solvent strength is
progressively raised. When compared to isocratic elution, gradient elution provided a shorter
total analysis while maintaining repeatability in retention time, peak area, peak height, and
linearity of the calibration curve. Moreover, some precautions need to be applied in this
experiment. In which, make sure the mobile phase composition, sample preparation, and
injection processes are optimised for each analysis, and that the HPLC equipment is kept up to
date.
 Compounds Structure No. of carbon
Caffeine 8
(1)

Acetone (2) 3

Methyl Benzoate (4) 8

Phenetole (3) 8

Phenanthrene (5) 14

Table 4: Structures of the compounds and the number of carbon contained in the
compounds.
CONCLUSION:

High performance liquid chromatography (HPLC) is a method of chromatography known for

its superior performance.Pressure was employed rather than gravity to divide mixture into
smaller components but needed to create a technique to demonstrate the durability of a blend
for future utilization. This experiment examines polarity.The analyte has been altered. Polarity
refers to the makeup of a mixture. Consequently, the optimal ratio of the blend is 70%
acetonitrile and 30% water. When the direction of the strength of the eluent rises with organic
solvent, leading to longer retention time reduces. The initial peak represents caffeine,with
subsequent peaks indicating other compounds present peak of acetone, methyl benzoate,
phenatole and phenanthrene.
REFERENCES:

Borai, E. H., Lasheen, Y. F., Seliman, A. F., & Aly, H. F. (2008). Gradient elution method for

successive separation of common cations and hydrophobic amines using suppressed ion
chromatography. Journal of liquid chromatography & related technologies, 31(6), 838-
849.

https://www.tandfonline.com/doi/abs/10.1080/10826070801891460

Joshi, V. S., Kumar, V., & Rathore, A. S. (2015). Role of organic modifier and gradient shape

in RP-HPLC separation: analysis of GCSF variants. Journal of chromatographic

science, 53(3), 417-423.

https://academic.oup.com/chromsci/article/53/3/417/290525?login=true

Today, C. (n.d.). The Role of Methanol and Acetonitrile as Organic Modifiers in

Reversed-phase Liquid Chromatography. Chromatography Today.

https://www.chromatographytoday.com/article/hplc-uhplc/31/advanced-
chromatography-technologies/the-role-of-methanol-and-acetonitrile-as-organic-
modifiers-in-reversed-phase-liquid-chromatography/2507

Taylor, T. (2022, April 15). Reversed-Phase HPLC Mobile-Phase Chemistry – Explained.

Chromatograph Online.

https://www.chromatographyonline.com/view/reversed-phase-hplc-mobile-phase-
chemistry-explained

L. (2021, November 13). 12.5: High-Performance Liquid Chromatography. Chemistry

LibreTexts.
https://chem.libretexts.org/Bookshelves/Analytical_Chemistry/Analytical_Chemistry_
2.1_(Harvey)/12%3A_Chromatographic_and_Electrophoretic_Methods/12.05%3A_H
 igh-
Performance_Liquid_Chromatography#:~:text=In%20reversed%2Dphase%20HPLC
%20the,mobile%20phase%20of%20lower%20polarity.

Is the caffeine molecule polar or non polar? | Socratic. (n.d.). Socratic.org.

https://socratic.org/questions/is-the-caffeine-molecule-polar-or-non-polar

Factors affecting resolution in HPLC. (n.d.).

https://www.sigmaaldrich.com/MY/en/technical-documents/technical-

article/analytical-chemistry/small-molecule-hplc/factors-affecting-resolution-in-hplc

HPLC Basics | Thermo Fisher Scientific - IE. (n.d.).

https://www.thermofisher.com/my/en/home/industrial/chromatography/chromatograp

hy-learning-center/liquid-chromatography-information/hplc-

basics.html#:~:text=High%2Dperformance%20liquid%20chromatography%20

Jandera, P. (2013). LIQUID CHROMATOGRAPHY | Normal phase. In Elsevier

eBooks. https://doi.org/10.1016/b978-0-12-409547-2.00300-0