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.

TLC vs. HPLC

(Diode Array) Detector 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

Varian Solvent Delivery System

Varian 9010 Solvent Delivery System 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

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

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

HPLC Chromatograms Area = base x height triangulation Absorbance  Approximation of peak area by triangulation Absorbance  Peak A Peak B 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

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

Normal vs. Reversed Phase Chromatography