chm 101/102 laboratory manual chromatography ii: hplc general chemistry 101/102 laboratory manual...

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



• 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



Hewlett-PackardHewlett-PackardSeries 1100 HPLCSeries 1100 HPLC

caffeinecaffeinewaterwater methanolmethanol(very polar) (polar) (fairly nonpolar)

CHM 101/102CHM 101/102



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



Hewlett-PackardHewlett-PackardSeries 1100 HPLCSeries 1100 HPLC

solventsolvent

pumppump

injectorinjector

columncolumn

detectordetector

CHM 101/102CHM 101/102



HPLC ColumnHPLC Column

CHM 101/102CHM 101/102



HPLC PumpHPLC Pump

CHM 101/102CHM 101/102



HPLC Autosampler and InjectorHPLC Autosampler and Injector

CHM 101/102CHM 101/102



HPLC DetectorHPLC Detector

UV/Visible SpectrophotometerUV/Visible Spectrophotometer

CHM 101/102CHM 101/102



HPLC Waste CollectionHPLC Waste Collection

CHM 101/102CHM 101/102



• 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



• 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



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



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



CHM 101/102CHM 101/102



• 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

chm 101/102 laboratory manual chromatography ii: hplc general chemistry 101/102 laboratory manual...

Documents

hplc slide

hplc hplc autosampler

hplc hplc column slide

hplc hplc pump slide

hplc sample

hplc vial

caffeine slide

hplc caffeine water