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Experiment 5Analysis of Caffeine in Beverages by High-
Performance Liquid Chromatography
(HPLC)
winter 2021
, Abstract
HPLC is a widely used technique for separation of components in a mixture. In this experiment,
HPLC was used to investigate the amount of caffeine in commercially available pops and energy
drink. The idea behind separating caffeine from these products was to perform elution on basis of
polarity and hence caffeine being the most polar, elutes first. After the determination of the
amount of caffeine in the unknowns, the result was compared to the recommended amount per
day that is 400mg. The amount of caffeine found in the unknowns were 39.8 mg and 65.3 mg.
Theory
High-performance liquid chromatography (HPLC) is a technique in analytical chemistry used to
separate the components in a mixture, and to identify and quantify each component.[1]
Instrumention
FIGURE. HPLC instrumentation. [2]
The Pump is situated in the most upper stream of the liquid chromatography framework and
creates a progression of eluent or more commonly known as the mobile phase.
The sample is introduced through the flow of the eluent by the injector.
The separation is performed inside the column, those are often made of stainless steel instead of
glass. The packing is generally silica or polymer gels.
After the separation of the analytes, a detector is used to observe the separation. The structure of
the eluent is predictable when no analyte is available. While the presence of analyte changes the
creation of the eluent. What detector does is to gauge these distinctions.
All these changes in the eluent and the whole system are recorded in form of electronic system.
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