Names: Lejaha Retŝepile
Student #:202101746
Co-workers: Manthipo Maqoacha (202101192)
Experiment #: 6
Experiment title: Determination of Parabens in a Standard Solution Using HPLC with Hypersil 5
CN Column and UV Detection at 260 nm
Course: Analytical Chemistry-C3620
Date of experiment: 04 December 2023
Date of submission: 18 December 2023
Objective(s):
The aim of this experiment is to determine the chromatograms (at 260 nm) of a 3mL standard
solution composed of a mixture of four parabens (methylparaben, ethylparaben, propylparaben,
and butylparaben) using HPLC.
Abstract:
The aim of the experiment is to determine the chromatograms at 260 nm of a 3 mL standard
solution composed of a mixture of four Parabens using HPLC with water: Acetonitrile (70:30) as
the mobile phase at pH 7. The Hypersil 5 CN column (150*4.6 mm) was employed as the
stationary phase. Peaks 1, 2, 3, and 4 were observed at 3.831, 5.994, 10.477, and 18.999 min,
respectively which are for methylparaben, ethylparaben, propylparaben, and butylparaben,
respectively. The retention times and masses of 0.01g for each paraben were used to prepare a
1000 ppm concentration for each. Results indicate efficient separation and identification of
parabens.
Introduction:
High-Performance Liquid Chromatography (HPLC) and Gas Chromatography (GC) are both
analytical techniques used for the separation of compounds [1]. HPLC uses a liquid mobile phase
and is suitable for analyzing soluble compounds [2]. GC, on the other hand, uses an inert or
unreactive gas as the mobile phase and is used for volatile compounds [3]. The mobile phases for
both HPLC and GC are prepared carefully to ensure accurate results [4]. to determine the
chromatograms (at 260 nm) of 3mL standard Solution composed of mixture of four Parabens
using HPLC water: Acetonitrile (70:30) as the mobile phase at pH 7 with the flow rate of 1.5
mL/min for 20 min. Hypersil 5 CN column with 150*4.6 mm dimensions was used for a
stationary phase.
Procedure:
The standard solution for HPLC was prepared carefully, ensuring accurate concentrations, A
1000 ppm concentration for each paraben (methylparaben, ethylparaben, propylparaben, and
butylparaben) was prepared by weighing 0.01g of each and diluting appropriately. The HPLC
instrument is then turned on and allowed to equilibrate and the sequence (start by powering on
system components like the pump and detector, then launch the Chromera software on the
computer. Configure parameters such as pump flow rate and column temperature. Prepare the
mobile phase and sample, prime the pump, and purge the system to ensure stability. Inject the
sample using the software or autosampler keypad, monitor the chromatogram, and system status.
Finally, process and report the data using the Chromera software for tasks like peak integration
and generating analysis reports. For detailed steps, refer to the provided sources, including
manuals and PerkinElmer's blog.) of operation was strictly followed. The method used in HPLC
analysis involved the careful injection of the standard solution into HPLC system with a Hypersil
5 CN column (150*4.6 mm) and monitoring of the resulting chromatogram. The mobile phase
used is a mixture of water and acetonitrile in a 70:30 ratio, at pH 7. The flow rate is set at 1.5
mL/min for a duration of 20 minutes. The stationary phase is a Hypersil 5 CN column with
dimensions of 150*4.6 mm. The separation of parabens was achieved, At the end of the run, the
flow is stopped, the sample is removed, and the instrument is turned off with the reverse order of
above-mentioned sequence.
Results:
Figure 1.HPLC results
Peaks 1, 2, 3, and 4 were observed at 3.831, 5.994, 10.477, and 18.999 min, respectively which
are for methylparaben, ethylparaben, propylparaben, and butylparaben, respectively.
Calculations:
approximating area of each peak
for peak 1
w(width) = 4.1-3.7 = 0.4
h(height) = 1070
1
A(area) = ×w×h
2
1
= × 0.4 × 1070 = 214
2
Peak Retention time(min) width height Area
1 3.831 0.4 1070 214
2 5.994 0.45 690 155.25
3 10.477 0.65 90 29.25
4 18.999 1.45 30 21.75
Discussion:
Methyl paraben produced a tailing peak (peak 1) at retention time of 3.831 minutes. each peak in
the chromatogram was carefully analyzed. The type of each peak was determined based on its
shape and other characteristics. The peaks were then labeled according to the analytes they
correspond to. The efficient separation and identification of parabens highlight the effectiveness
of the HPLC method under the described conditions.
Conclusion:
The aim of the experiment was successfully achieved. The HPLC analysis provided results
which shows that the experiment successfully determined the chromatograms of a standard
solution containing four parabens using HPLC. The method provides a reliable means for
separating and identifying these compounds in a mixture.
References:
[1]: Skoog Fundamental of Analytical Chemistry,9E, G-14
[2]: Understanding Chromatogram Peaks - Chemtech International.
https://chemtech-us.com/articles/understanding-chromatogram-peaks-fronting-tailing-ghosting-
rounded-explained/.
[3]: High Performance Liquid Chromatography - Chemistry LibreTexts.
https://chem.libretexts.org/Bookshelves/Analytical_Chemistry/Supplemental_Modules_
%28Analytical_Chemistry%29/Instrumentation_and_Analysis/Chromatography/
High_Performance_Liquid_Chromatography.
[4]: Gas Chromatography- Definition, Principle, Parts, Steps, Uses.
https://microbenotes.com/gas-chromatography/.
