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Experiment 6: High Performance Liquid Chromatography (HPLC)

This document describes an experiment using high performance liquid chromatography (HPLC) to analyze caffeine content. The objectives are to determine the retention time of a caffeine standard, identify the caffeine peak in soft drinks, and quantify the amount of caffeine using a response factor method. The procedure involves preparing caffeine standard solutions, filtering soft drink samples, running the standards and samples on an HPLC to measure retention times and peak areas for caffeine.

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27 views

Experiment 6: High Performance Liquid Chromatography (HPLC)

This document describes an experiment using high performance liquid chromatography (HPLC) to analyze caffeine content. The objectives are to determine the retention time of a caffeine standard, identify the caffeine peak in soft drinks, and quantify the amount of caffeine using a response factor method. The procedure involves preparing caffeine standard solutions, filtering soft drink samples, running the standards and samples on an HPLC to measure retention times and peak areas for caffeine.

Uploaded by

enieynaz
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PPTX, PDF, TXT or read online on Scribd
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EXPERIMENT 6

HIGH PERFORMANCE
LIQUID
CHROMATOGRAPHY
(HPLC)

OBJECTIVES
To determine the retention time of a
standard solution (caffeine)
To identify the caffeine peak in a soft
drink sample
To determine the amount of caffeine
in soft drink sample using the
response factor method

APPARATUS CHEMICALS

Beaker
Burette
Glass rod
Volumetric flask
10mL
Dropper

Caffeine
standard
Soft drink (coke
& revive)
Distilled water
Acetonitrile

PROCEDURE
1. Caffeine Standard Solutions.
i. Stock solution, 1.0 mg/mL (Solution A)
a) Weighed 10 mg caffeine accurately into a
10mL
volumetric flask.
b) Dissolved in distilled water.
c) The volume made up with distilled water.
ii. Working solution, 0.1 mg/mL (Solution B).
a) 1 mL of solution A has been pipette into a
10 mL volumetric flask.
b) The volume made up with distilled water.

2. Sample preparation.
i. About 10 mL of soft drink sample (coke & revive)
poured into a small beaker.
ii. A magnetic stirrer used to remove any
carbonation ( sonnicate if necessary).
iii. 2-5 mL aliquot filtered through a 0.45 m pore
diameter membrane filter to remove particulate
matter.
3. 10 L of solution B injected into the HPLC to
establish the retention time, tR, of caffeine.
4. The peak area of caffeine measured.
5. 10 L of the prepared soft drink injected into HPLC
instrument.
6. The peak area measured corresponding to caffeine
by referring to its tR.

HPLC INSTRUMENT

DATASHEET

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