CHM 101/102

Chromatography II: HPLC

General Chemistry 101/102

Laboratory Manual
University of North Carolina at Wilmington

Laboratory Manual
 CHM 101/102

Chromatography II: HPLC

• Purpose
 Learn the basic techniques of Hi-Performance
Liquid Chromatography (HPLC).
 Study the role played by intermolecular forces in
HPLC.

• Safety Considerations
 There are no special safety considerations or
waste disposal concerns associated with this
laboratory exercise.

Laboratory Manual
 CHM 101/102

Chromatography II: HPLC

Hewlett-Packard
Series 1100 HPLC

water methanol caffeine

(very polar) (polar) (fairly nonpolar)

Laboratory Manual
 CHM 101/102

Chromatography II: HPLC

HPLC Column
solvent
(mobile phase) to detector
and
sample
wax coated beads

nonpolar
stationary phase

microscopic view of bead

3 m
Laboratory Manual
 CHM 101/102

Chromatography II: HPLC

Hewlett-Packard
Series 1100 HPLC

solvent

pump

injector

column

detector

Laboratory Manual
 CHM 101/102

Chromatography II: HPLC

HPLC Column

Laboratory Manual
 CHM 101/102

Chromatography II: HPLC

HPLC Pump

Laboratory Manual
 CHM 101/102

Chromatography II: HPLC

HPLC Autosampler and Injector

Laboratory Manual
 CHM 101/102

Chromatography II: HPLC

HPLC Detector
UV/Visible Spectrophotometer

Laboratory Manual
 CHM 101/102

Chromatography II: HPLC

HPLC Waste Collection

Laboratory Manual
 CHM 101/102

Chromatography II: HPLC

• Procedure
 Use a 10 mL volumetric pipet to add 10.00 mL of soft
drink to 10.00 mL of deionized water.
 Mix thoroughly and half fill a HPLC vial with your
sample. Label the vial with your name and the name of
the soft drink.

Laboratory Manual
 CHM 101/102

Chromatography II: HPLC

• Analysis of Results
 Use the four caffeine standards to prepare a
calibration curve (graph) that plots peak area vs.
concentration.
 Draw a “best fit” straight line on your graph.
 Use your calibration curve to determine the
concentration of caffeine in the sample you prepared.
 Use the concentration of caffeine in your sample,
along with the dilution equation, to determine the
concentration of caffeine in the soft drink you used.

Laboratory Manual
 CHM 101/102

Chromatography II: HPLC

HPLC Chromatogram of Standard 1

0.500 x 10-4 M caffeine

Laboratory Manual
 CHM 101/102

Chromatography II: HPLC

HPLC Chromatogram of Standard 2

1.00 x 10-4 M caffeine

Laboratory Manual
 CHM 101/102

Chromatography II: HPLC

Laboratory Manual
 CHM 101/102

Chromatography II: HPLC

• Calculations
Remember, the original soft drink was diluted to prepare your HPLC
sample. The concentration (M) of caffeine in the original soft drink can be
calculated by using the dilution equation:

M1V1 = M2V2

M1V1
M2 =
V2

The concentration (mg caffeine / 500 mL soda) of caffeine in a full can of

your soft drink can be determine using the following equation:

M2 x 194.2 g caffeine x 1000 mg x 0.500 L = mg caffeine

mol caffeine g in bottle

Laboratory Manual