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EXPERIMENT 9
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
A. PURPOSE
The principal purpose of this experiment is to introduce you to procedures and
instrumentation used for high performance liquid chromatography (HPLC).
B. OVERVIEW
In this experiment you will separate mixtures of components frequently found in
soft drinks, namely caffeine, benzoate, and aspartame. You will be given seven solutions
containing known amounts of the three species and record the chromatograms.
C. INSTRUMENTATION
1. Liquid chromatograph
A commercially available liquid chromatograph (PM-80 pump and CC-5 Injector,
Bioanalytical Systems, West Lafayette, IN) is used in this experiment where separation
is performed on a reversed phase column (#MF 8954).
Chromatograph and column settings are as follows:
• Flow rate: 0.5 mL/min
• Minimum pressure setting: 250 PSI
• Maximum pressure setting: 4000 PSI
• Column dimensions: 3(i.d.) × 100 mm
• Column has silica packing (3µm particle size) and is functionalized with ODS
(C18). pH range is 2-7.
2. Detector
Detection is by absorption spectroscopy. Although the detector (Model UVVIS
200, Linear) is capable of measuring several wavelengths, all the components of interest
absorb at 254 nm. Accordingly, only one wavelength, 254 nm, will be used in this study.
Detector settings are as follows:
• Rise time: 0.3 s
• Wavelength: 254 nm
• Range: 0.2 AUFS (absorbance units full scale)
3. Data acquisition
Data will be recorded on line using a digital voltmeter and a digital computer. The
following settings should be used:
• Time per point: 0.1 s
• Time per chromatogram: 10 min initially
4. Saving data
Data will be saved on floppy disks. Each group should bring at least two
floppy disks with full storage capability free of other files.
D. SOLUTIONS
1. Solutions provided
a. Mobile phase (Eluent). The mobile phase (eluent) is prepared by mixing 400 mL
of acetonitrile with 2.3 mL of glacial acetic acid and diluting the solution to 2 L
with water. The resulting solution is then adjusted to pH = 4.2 by addition of
sodium hydroxide. The solution is then filtered through a 0.2 µm Nylon 66
membrane filter under vacuum to degas the solution and to remove solids that
SEPARATION OF COMPONENTS OF SOFT DRINKS
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