FACULTY OF APPLIED SCIENCES

CHM 571 - BASIC INSTRUMENTAL ANALYSIS

LAB REPORT

NAME: MOHAMAD SAIFUL BIN MOHD RAFFIAH

STUDENT ID: 2021459156
CLASS GROUP: AS239 4A
EXPERIMENT 3: HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
(HPLC)
LECTURER: DR. NORAINI BINTI KASIM
DATE OF EXPERIMENT: 10th MAY 2023
DATE OF SUBMISSION: 14th JULY 2023
Tittle : High Performance Liquid Chromatography (HPLC)

Objective
1. To determine the retention times of naphthalene and anthracene solutions
2. To determine the composition of an unknown amount of a sample mixture with unknown
quantity of the two components using the following methods:
i. area percent
ii. external standard

Materials
Standard Mixture : naphthalene = 0.246 mg/mL
anthracene = 0.115 mg/mL

Unknown sample
All sample are dissolved in mobile phase solution

Instrument
Perkin Elmer with UV detector

Introduction
High-performance liquid chromatography (HPLC) is a technique for separating, identifying,
and quantifying components in a mixture. It is the primary chromatography technique utilised in
the majority of laboratories across the world. Under gravity, a solvent drips through a column
loaded with an adsorbent in column chromatography. HPLC is an advanced type of column
chromatography. Under high pressures of up to 400 atmospheres, a pump drives a solvent
through a column. The granular material of solid particles such as silica or polymers is often used
as the column packing material, adsorbent, or stationary phase.

When compared to column chromatography, the pressure makes the process much faster.
This enables the use of much smaller particles as column packing material. Smaller particles have
a substantially larger surface area for interactions between the stationary phase and the
molecules passing by. This leads in a significantly better separation of the mixture's components.
Because of their varying degrees of interaction with the absorbent particles, the components of a
mixture are separated from one another. This results in varying elution rates for the various
components, resulting in separation of the components as they flow out the column. When
compared to column chromatography, HPLC is more automated and sensitive.
Procedures
1. In this experiment, the column RP18 was used in the HPLC instrument, and portion of
acetonitrile: water (70: 30) (v/v) used as mobile phase. Then, the instrument was switch on and
set the pressure to 200 psi and the flow rate was 2mL/ min. The wavelength of the UV detector
was 254 nm.
2. Using a syringe to introduce naphthalene into the sampling loop in the load mode. After the
sample was introduced, turn it into the inject mode. Then, the retention time and the area
component were recorded.
3. The procedure was repeated with anthracene solution, standard mixture and sample solution.
All of the results were recorded and observed.
4. The solvent reservoir is a mobile phase content. The mobile phase in HPLC is a mixture of
polar and non-polar liquid components such as acetonitrile: water (70: 30) (v/v). The stationary
phase for HPLC is naphthalene, anthracene, standard mixture and unknown solution.
5. The pump used to force the mobile phase to flow through the packing and to maintain stable
flow. The sample injector should provide injection of the liquid sample within the range of 0.5 -
5 L of volume with high reproducibility and under high pressure to 7000 psi.
6. The column is stainless steel, are between 50 and 300 mm long and have an internal diameter
of between 2 and 5 mm. Columns with internal diameters of less than 2 mm are often referred
to as microbore columns. Detector is located at the end of the column detect the analytes as
they elute from the chromatographic column.
7. The amount of light absorbed will depend on the amount of a particular compound that is
passing through the beam at the time. Signals from the detector were collected and integrates
into chromatograph that can be read.
Results
Data

Retention time (min) Average Peak area (cm2) Average

Sample
1 2 (min) 1 2 (cm2)
100.0000 100.0000
Naphthalene 1.531 1.531 1.531 100 100 1.0000
= 1.0000 = 1.0000
100.0000 100.0000
Anthracene 1.947 1.948 1.948 100 100 1.0000
= 1.0000 = 1.0000
9.1783 9.2170
1.533 1.533 1.533 100 100 0.0920
Standard = 0.091783 = 0.09217
Mixture 90.8217 90.7830
1.951 1.949 1.950 100 100 0.9080
= 0.908217 = 0.90783
12.4098 12.4088
1.532 1.533 1.533 100 100 0.1241
Unknown = 0.124098 = 0.124088
Mixture 87.5902 87.5912
1.949 1.951 1.950 100 100 0.8756
= 0.875902 = 0.875912
Retention Time (min) of compounds

!.!#$% ' !.!((+ '

Naphthalene : x 1000 = 0.246 mg/mL Anthracene : x 1000 = 0.115 mg/mL
(!! )* (!! )*
Calculation
,-./ 01-.
Response Factor =
2345-461.6734
Naphthalene
(.!!!!
RF = = 4.0650
!.#$%

Anthracene
(.!!!!
RF = = 8.6957
!.((+

Standard mixture
!.!8#!
RF(Rt1) = = 0.3740
!.#$%

!.8!9!
RF(Rt2) = = 7.8957
!.((+

Unknown mixture
!.(#$(
Conc.(Rt1) = = 0.3310
!.:;$8

!.9;+%
Conc.(Rt2) = = 0.1109
;.98+;

!.(#$(
RF(Rt1) = = 0.3749
!.::(!

!.9;+%
RF(Rt2) = = 7.8954
!.((!8
Discussion
1. Draw the structural formula of naphthalene and anthracene.

Naphthalene Anthracene
2. Using the values of the standard mixture, calculate the response factor for naphthalene and
anthracene.
Naphthalene
(.!!!!
RF = = 4.0650
!.#$%

Anthracene
(.!!!!
RF = = 8.6957
!.((+
3. Calculate the amount (mg/mL) of each component in the sample mixture.
!.!8#!
RF(Rt1) = = 0.3740
!.#$%

!.8!9!
RF(Rt2) = = 7.8957
!.((+
4. Identify the type of HPLC employed.
- In HPLC, the analysis was carried out using normal phase against reverse phase. The
separation is regarded as normal phase if the stationary phase is more polar than the mobile
phase. The separation is the reverse phase if the stationary phase is less polar than the mobile
phase. The reverse phase will result in a longer retention period.
5. What is the detector used for the sample ?
- HPLC detector
6. What is the detector used ?
- To identify mixture elements that are eluting from the chromatography column. As the
components leave (elute) the column, the detector detects their presence.
7. What is the difference between isocratic and gradient elution ? Which type of elution was used
in this experiment ?
- According to the isocratic method, mobile phase's combination must remain constant
throughout the whole testing period. By using a gradient, it is implied that the eluent mixture
becomes more concentrated and charged during measurement, which affects the retention of
analytes. Either the separation can be sped up or slowed down. The most thorough separation
of the peak is provided by gradient elution, which can be done quickly.

Conclusion
To conclude, the retention time for the naphthalene is 1.531. For anthracene, the retention time
is 1.947, for standard mixture there are two retention times that are 1.5330 and 1.9500. For the
unknown solution the retention time is 1.5330 and 1.9500. The standard mixture results show that
the retention time for the naphthalene is faster than anthracene because the naphthalene elutes
faster in the mobile phase. Naphthalene is the most polar component in the standard mixture.
The unknown solution also shows that the naphthalene appears faster than anthracene. This was
!"#$ &'"#
determined by using the formula of response factor, RF = . The average
()*+"*,'#,-)*
2
naphthalene and anthracene peak area was the same which was 1.000 cm . Besides, unknown
mixture and standard mixture was shown two values for each because it contains two
components. The retention time in the unknown mixture and standard mixture can be determined
on which peak by calculating the response factor. So, the results obtained stated that peak 1 on
unknown mixture and standard mixture was naphthalene followed by anthracene on peak 2.
Hence, Naphthalene is the most polar component in the standard mixture.

Reference

1. High Performance Liquid Chromatography (HPLC) | ELGA LabWater. (n.d.).

Www.elgalabwater.com. https://www.elgalabwater.com/high-performance-liquid-
chromatography
2. Bower, P. (2023). High-Performance Liquid Chromatography (HPLC) | Protocol.
Www.jove.com. https://www.jove.com/v/10156/high-performance-liquid-
chromatography-hplc