Organic Chemistry I - Lab

1. Organic Chemistry I - Lab High Performance Liquid Chromatography HPLC is characterized by the use of high pressure to push a mobile phase solution through a column of stationary phase allowing separation of complex mixtures with high resolution.

2. TLC vs. HPLC

3. Varian HPLC System 9060 Polychrom (Diode Array) Detector Computer Workstation 9050 Variable UV/Vis Detector 9010 Solvent Delivery System HPLC Solvent Reservoirs Rheodyne Injector Keep an eye on these 4 screens! HPLC Column

4. Varian Solvent Delivery System

5. to column %A %B %C Flow Rate Pressure {H2O} {MeOH} (mL/min) (atmos.) load Ready inject Rheodyne Injector Varian 9010 Solvent Delivery System to injector through pump Column C through pulse dampener A B from solvent reservoir to detector Ternary Pump

6. Variable UV/Vis Detector ABS AUFS l RunTime EndTime 0.001 2.000 238 0.00 min 10.0 min Ready

7. Wavelength Time Varian 9060Polychrom Detector Ready UV Spectrum {shows full UV abs.} UV Spectrum UVmax UVmax Chromatogram ABS. Reset Rt Rt ABS. Chromatogram {shows peaks, Rt}

8. HPLC Chromatograms Approximation of peak area by triangulation Peak A Peak B Absorbance  height 0 1 2 3 4 5 6 7 Time (minutes) base Rt = 3.0 min. faster moving less retained Rt = 5.2 min. slower moving more retained Area =base x height 2

9. Derivatized Silica Gel Where R = C18H37 hydrocarbon chain (octadecylsilyl deriv. silica or “C18”) bulk (SiO2)x surface bulk (SiO2)x surface Chromatography Stationary Phases Silica Gel relatively polar surface relatively nonpolar surface “normal phase” “reversed phase”

10. Normal vs. Reversed Phase Chromatography