1. Chromatography II: HPLC
General Chemistry 101/102 Laboratory Manual University of North Carolina at Wilmington

2. 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.

3. Hewlett-Packard Series 1100 HPLC
Chromatography II: HPLC
Hewlett-Packard Series 1100 HPLC
caffeine
methanol
water
(very polar)
(polar)
(fairly nonpolar)

4. Chromatography II: HPLC
HPLC Column
solvent (mobile phase) and sample
to detector
wax coated beads
nonpolar stationary phase
microscopic view of bead
3 mm

5. Hewlett-Packard Series 1100 HPLC
Chromatography II: HPLC
Hewlett-Packard Series 1100 HPLC
solvent
pump
injector
column
detector

6. Chromatography II: HPLC
HPLC Column

7. Chromatography II: HPLC
HPLC Pump

8. HPLC Autosampler and Injector
Chromatography II: HPLC
HPLC Autosampler and Injector

9. UV/Visible Spectrophotometer
Chromatography II: HPLC
HPLC Detector
UV/Visible Spectrophotometer

10. Chromatography II: HPLC
HPLC Waste Collection

11. Chromatography II: HPLC
Procedure
Use a 10 mL volumetric pipet to add mL of soft drink to 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.

12. 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.

13. HPLC Chromatogram of Standard 1
Chromatography II: HPLC
HPLC Chromatogram of Standard 1
0.500 x 10-4 M caffeine

14. HPLC Chromatogram of Standard 2
Chromatography II: HPLC
HPLC Chromatogram of Standard 2
1.00 x 10-4 M caffeine

15. Chromatography II: HPLC

16. 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 V2
M1V1
M2 =
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 g caffeine x mg x L = mg caffeine
mol caffeine g
in bottle