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Chromatography II: HPLC

Chromatography II: HPLC. General Chemistry 101/102 Laboratory Manual University of North Carolina at Wilmington. Purpose. Learn the basic techniques of Hi-Performance Liquid Chromatography (HPLC). Study the role played by intermolecular forces in HPLC. Safety Considerations.

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Chromatography II: HPLC

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

  2. 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. Chromatography II: HPLC

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

  4. 3 mm Chromatography II: HPLC HPLC Column solvent(mobile phase)andsample to detector wax coated beads nonpolarstationary phase microscopic view of bead

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

  6. Chromatography II: HPLC HPLC Column

  7. Chromatography II: HPLC HPLC Pump

  8. Chromatography II: HPLC HPLC Autosampler and Injector

  9. 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 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.

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

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

  15. Chromatography II: HPLC

  16. 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 in bottle g mol caffeine Chromatography II: HPLC

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