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Analysis of Caffeine using HPLC
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it is a experimental report with full procedure, results, calculation and discussion of the results with the findings of the aim of the experiment
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Surname: Ragolane KCTitle: High performance liquid
chromatography
Experimental date:01 February
2021
Aim
To determine the caffeine concentration of an anadin tablet sample using High Perfomannce Liquid
, Chromatography
Abstract
Concentrated standards of each salt of Aspirin, caffeine and paracentamol were prepared and the
concentrations needed for the preparation of the standards of each salt were determined. Mixture T
prepared from each stock solution was used for the optimization of chromatographic conditions to
determine the effect of the instrumental parameters to give the best resolution of the chromatogram
and good separations .The parameters used were ; a flow rate of 0.8 mL/min ,wavelength of 220 nm
and the column was washed with acetonitrile: water (60:40 v/v ).The anadin sample tablet was
weighed and it was prepared for analysis with a 25 times dilution included .
The standards and the sample were injected then analyzed on a HPLC instrument . The chromatogram
results were obtained which were used to plot a linear curve for the determination of the exact
concentration of caffeine which was found to be 52.94 %.Different peaks were seen on the
chromatogram of the sample ,to identify the peak of the analyte(caffeine) in our sample the retention
time of each component were compared to that of the sample peaks .
Introduction
High performance liquid chromatography is a commonly used separation technique used to separate a
mixture of compounds it is mostly used throughout the chemical, biochemical, biological and
pharmaceutical sciences so as to identify or quantify individual components of a mixture. some
application include drug analysis ,water purification , detection of impurities ,determining amount of
aspirin the for pharmaceutical industries and determination of caffeine in beverages in the food
industries .The are several types of liquid chromatography but the main one focused in this experiment
is the high pressure liquid chromatography(HPLC), the HPLC separates compounds that are dissolved
in a solution and they are separated by injecting a small amount of the sample into a column, The
components pass through the column at different rates due to the partitioning of the samples [2] .
Qualitative analysis is possible by injection of standards and comparison of the retention times
obtained, quantitative determination of a particular component can be achieved by analyzing the peak
height or peak area .HPLC is a form of column chromatography , but instead of using gravity , HPLC uses
pressure to force the mobile phase through the column leading to faster separations [4] .In small
amounts of approximately 20 μL ,the sample mixture to be separated and analyzed is sent into a stream
of mobile phase then into the column, the mixture moves through the column at varying velocities and
interacts with the sorbent known as the stationery phase, the speed of each component depends on its
chemical nature , the nature of the column and the composition of the mobile phase .The commonly
used mobile phases include water and organic solvents such as acetonitrile or methanol [3].
It has a major advantage over gas chromatography since it doesn’t require compounds to be volatile in
which only 15% of most compounds fit these requirements. In HPLC we need compounds that are
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