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HPLC
HPLC
Introduction
Instrument
Materials
Column : RP 18
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
Results
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
analysis.
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.
According to table above, the average retention time of standard Naphtalene, 1.930, is
faster than standard Anthracene, 3.014.
peak area( )
x 100
total peak area( )
Total area:
24.89 + 64.17
= 89.06
Mole % 1:
24.89
x 100
89.06
= 27.95%
Mole % 2:
24.89 64.17
x 100
89.06
= 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:
(Cm2)
= 1956.6
= 1681.6
= 823
0.5038 0. 5038
0.5/ 1000
= 1007.8
= 497.8
0.6417 0. 6417
0.5/ 1000
= 1283.4
Conclusion
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.
Reference
Barkovich , M. (n.d.). Chemistry LibreTexts. Retrieved November 11, 2017, from High performance liquid
chromatography:
https://chem.libretexts.org/Core/Analytical_Chemistry/Instrumental_Analysis/Chromatography/
High_performance_liquid_chromatography
Clark, J. (2007). Retrieved November 11, 2017, from high performance liquid chromatography - hplc:
https://www.chemguide.co.uk/analysis/chromatography/hplc.html
Giri, D. (2015, July 28). Laboratoryinfo.com. Retrieved November 11, 2017, from High Performance
Liquid Chromatography (HPLC) : Principle, Types, Instrumentation and Applications:
http://laboratoryinfo.com/hplc/