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CHM 101/102CHM 101/102
Laboratory ManualLaboratory ManualLaboratory Manual
Chromatography II: HPLCChromatography II: HPLC
General Chemistry 101/102General Chemistry 101/102Laboratory ManualLaboratory Manual
University of North Carolina at WilmingtonUniversity of North Carolina at Wilmington
CHM 101/102CHM 101/102
Laboratory ManualLaboratory ManualLaboratory Manual
Chromatography II: HPLCChromatography II: HPLC
• PurposePurpose Learn the basic techniques of Hi-Performance Learn the basic techniques of Hi-Performance
Liquid Chromatography (HPLC).Liquid Chromatography (HPLC). Study the role played by intermolecular forces in Study the role played by intermolecular forces in
HPLC.HPLC.
• Safety ConsiderationsSafety Considerations There are no special safety considerations or There are no special safety considerations or
waste disposal concerns associated with this waste disposal concerns associated with this laboratory exercise.laboratory exercise.
CHM 101/102CHM 101/102
Laboratory ManualLaboratory ManualLaboratory Manual
Chromatography II: HPLCChromatography II: HPLC
Hewlett-PackardHewlett-PackardSeries 1100 HPLCSeries 1100 HPLC
caffeinecaffeinewaterwater methanolmethanol(very polar) (polar) (fairly nonpolar)
CHM 101/102CHM 101/102
Laboratory ManualLaboratory ManualLaboratory Manual
Chromatography II: HPLCChromatography II: HPLC
solventsolvent((mobile phasemobile phase))
andandsamplesample
wax coated beadswax coated beads
HPLC ColumnHPLC Column
nonpolarnonpolarstationary phasestationary phase
to detectorto detector
3 m
microscopic view of beadmicroscopic view of bead
CHM 101/102CHM 101/102
Laboratory ManualLaboratory ManualLaboratory Manual
Chromatography II: HPLCChromatography II: HPLC
Hewlett-PackardHewlett-PackardSeries 1100 HPLCSeries 1100 HPLC
solventsolvent
pumppump
injectorinjector
columncolumn
detectordetector
CHM 101/102CHM 101/102
Laboratory ManualLaboratory ManualLaboratory Manual
Chromatography II: HPLCChromatography II: HPLC
HPLC ColumnHPLC Column
CHM 101/102CHM 101/102
Laboratory ManualLaboratory ManualLaboratory Manual
Chromatography II: HPLCChromatography II: HPLC
HPLC PumpHPLC Pump
CHM 101/102CHM 101/102
Laboratory ManualLaboratory ManualLaboratory Manual
Chromatography II: HPLCChromatography II: HPLC
HPLC Autosampler and InjectorHPLC Autosampler and Injector
CHM 101/102CHM 101/102
Laboratory ManualLaboratory ManualLaboratory Manual
Chromatography II: HPLCChromatography II: HPLC
HPLC DetectorHPLC Detector
UV/Visible SpectrophotometerUV/Visible Spectrophotometer
CHM 101/102CHM 101/102
Laboratory ManualLaboratory ManualLaboratory Manual
Chromatography II: HPLCChromatography II: HPLC
HPLC Waste CollectionHPLC Waste Collection
CHM 101/102CHM 101/102
Laboratory ManualLaboratory ManualLaboratory Manual
Chromatography II: HPLCChromatography II: HPLC
• ProcedureProcedure Use a 10 mL volumetric pipet to add 10.00 mL of soft Use a 10 mL volumetric pipet to add 10.00 mL of soft
drink to 10.00 mL of deionized water.drink to 10.00 mL of deionized water. Mix thoroughly and half fill a HPLC vial with your Mix thoroughly and half fill a HPLC vial with your
sample. Label the vial with your name and the name of sample. Label the vial with your name and the name of the soft drink.the soft drink.
CHM 101/102CHM 101/102
Laboratory ManualLaboratory ManualLaboratory Manual
Chromatography II: HPLCChromatography II: HPLC
• Analysis of ResultsAnalysis of Results Use the four caffeine standards to prepare a Use the four caffeine standards to prepare a
calibration curve (graph) that plots peak area vs. calibration curve (graph) that plots peak area vs. concentration.concentration.
Draw a “best fit” straight line on your graph.Draw a “best fit” straight line on your graph. Use your calibration curve to determine the Use your calibration curve to determine the
concentration of caffeine in the sample you prepared.concentration of caffeine in the sample you prepared. Use the concentration of caffeine in your sample, Use the concentration of caffeine in your sample,
along with the dilution equation, to determine the along with the dilution equation, to determine the concentration of caffeine in the soft drink you used.concentration of caffeine in the soft drink you used.
CHM 101/102CHM 101/102
Laboratory ManualLaboratory ManualLaboratory Manual
Chromatography II: HPLCChromatography II: HPLC
HPLC Chromatogram of Standard 1HPLC Chromatogram of Standard 1
0.500 x 100.500 x 10-4-4 M caffeine M caffeine
CHM 101/102CHM 101/102
Laboratory ManualLaboratory ManualLaboratory Manual
Chromatography II: HPLCChromatography II: HPLC
HPLC Chromatogram of Standard 2HPLC Chromatogram of Standard 2
1.00 x 101.00 x 10-4-4 M caffeine M caffeine
CHM 101/102CHM 101/102
Laboratory ManualLaboratory ManualLaboratory Manual
Chromatography II: HPLCChromatography II: HPLC
CHM 101/102CHM 101/102
Laboratory ManualLaboratory ManualLaboratory Manual
Chromatography II: HPLCChromatography II: HPLC
• CalculationsCalculationsRemember, 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
M1V1M2 =
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
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