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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.
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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.
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
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
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}
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
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”