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1. To determine the retention times of naphthalene and anthracene solutions.

2. To determine the composition of an unknown amount of a mixture containing the two
components using the following methods.
 Normalization method
 External standard method
 Area percent
3. To calculate the response factor and then determine the concentration of each
component in the sample mixture.


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 which is a solid, or a liquid supported on a solid and a
mobile phase which is a liquid or a gas. The mobile phase flows through the stationary
phase and carries the components of the mixture with it.

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.
There are following variants of HPLC, depending upon the phase system (stationary) in
the process:

1. Normal Phase HPLC:

This 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.
2. 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.


785A UV/VIS Detector – HPLC


1. Standard Naphthalene solution : 0.5g/1000 mL

2. Standard Anthracene : 0.5/1000 mL
3. Standard Mixture
4. Unknown Solution

All samples are dissolved in mobile phase solution

Experimental procedure:

Column : RP 18

Mobile phase : Acetonitrile : Water

70% : 30% (v/v)

The instrument was switched on

4 sample is used which are Pressure : 350 bar

standard naphthalene, standard
Flow rate : 2.0 mL/min
Arthracene, standard mixture and
unknown mixture.

The sample was sucked into Then, the sample in the The signal from detector was
a syringe syringe was injected into the printed out
injector of HPLC

Sample Retention Time Time

tR1 tR2 tR3
Standard Napthalene 1.996 1.813 1.982 1.930
Standard Anthracene 3.010 3.027 3.005 3.014
Standard Mixture 2.003 1.939 1.985 1.976
3.020 2.994 3.003 3.006
Unknown Mixture 1.987 1.990 1.977 1.985
3.042 3.025 3.006 3.024
Table 1: Retention time (min) of compounds

Sample Peak Area (cm2) Response

Peak Area Peak Area Peak Area Peak
1 2 3 Average
Standard 0.9799 0.9794 0.9756 0.9783 1956.6
Standard Anthracene 0.8484 0.8732 0.8007 0.8408 1681.6
Standard Mixture 0.3721 0.4311 0.4313 0.4115 823
0.5370 0.4827 0.4918 0.5038 1007.6
Unknown Mixture 0.2393 0.2560 0.2513 0.2489 497.8
0.6635 0.6267 0.6349 0.6417 1283.4
Peak area (cm ) of samples

As shown in the schematic diagram in Figure above, HPLC instrumentation includes a

pump, injector, column, detector and integrator or acquisition and display system. The
heart of the system is the column where separation occurs.

In Solvent Reservoir, Mobile phase contents are contained in a glass reservoir. The
mobile phase in HPLC is usually a mixture of polar and non-polar liquid components.
Then, a pump aspirates the mobile phase from the solvent reservoir and forces it
through the system’s column and detector. After that the sample will went through an
injector. The sample injector can be a single injection or an automated injection system.
An injector for an HPLC system should provide injection of the liquid sample with high
reproducibility and under high pressure.

HPLC columns are usually made of polished stainless steel, are between 50 and 300
mm long and have an internal diameter of between 2 and 5 mm. They are commonly
filled with a stationary phase with a particle size of 3–10 µm. Columns with internal
diameters of less than 2 mm are often referred to as microbore columns. Ideally the
temperature of the mobile phase and the column should be kept constant during an

The HPLC detector is located at the end of the column detect the analytes as they elute
from the chromatographic column. Commonly used detectors are UV-spectroscopy,
fluorescence, mass-spectrometric and electrochemical detectors.

Lastly, the Signals from the detector may be collected on chart recorders or electronic
integrators that vary in complexity and in their ability to process, store and reprocess
chromatographic data. The computer integrates the response of the detector to each
component and places it into a chromatograph that is easy to read and interpret.

The retention times Naphthalene and Anthracene

Retention time is the time taken for a particular compound to travel through the column
to the detector. This time is measured from the time at which the sample is injected to
the point at which the display shows a maximum peak height for that compound.
Different compounds have different retention times. For a particular compound, the
retention time will vary depending on the pressure, the nature of the stationary phase,
which is its material and particle size, the exact composition of the solvent and the
temperature of the column.

Samples Average Retention Time, tR

Standard Naphthalene 1.930
Standard Anthracene 3.014

According to table above, the average retention time of standard Naphtalene, 1.930, is
faster than standard Anthracene, 3.014.

Composition of an unknown amount of a mixture containing the two components

Composition of an unknown amount of a mixture containing two components can be
determined by using this following formula:

The percentage for each of composition of unknown sample:

peak area( )
x 100
total peak area( )
Total area:

24.89 + 64.17

= 89.06

Mole % 1:

x 100

= 27.95%

Mole % 2:

24.89 64.17
x 100

= 72.05%

Response Factor

The ratio between the concentration of a compound being analysed and the response of
the detector to that compound is known as response factor. A chromatography will show
a response from a detector as a peak. The response factor can be calculated by using
the following formula:

Response Factor = Peak Area / Concentration

Samples Response Factor
Average Peak Area


Standard Naphthalene 0.9783 0.9783

0.5/ 1000

= 1956.6

Standard Anthracene 0.8408 0. 8408

0.5/ 1000

= 1681.6

Standard Mixture 0.4115 0.4 115

0.5/ 1000

= 823

0.5038 0. 5038
0.5/ 1000

= 1007.8

Unknown Mixture 0.2489 0. 2489

0.5/ 1000

= 497.8

0.6417 0. 6417
0.5/ 1000

= 1283.4
By running this experiment, the system of HPLC may be understood thoroughly and the
retention time of sample could be determined. The retention time of standard
naphthalene, standard Anthracene, are 0.948, and 0.978 respectively. Last but not
least, the composition of unknown amount mixture containing the two compositions is
also can be determined which are 27.95% and 72.05% respectively.

Barkovich , M. (n.d.). Chemistry LibreTexts. Retrieved November 11, 2017, from High performance liquid

Clark, J. (2007). Retrieved November 11, 2017, from high performance liquid chromatography - hplc:

Giri, D. (2015, July 28). Retrieved November 11, 2017, from High Performance
Liquid Chromatography (HPLC) : Principle, Types, Instrumentation and Applications: