Organic Chemistry I - LabHigh Performance Liquid ChromatographyHPLC is characterized by the use of high pressure to push amobile phase solution through a column of stationary phaseallowing separation of complex mixtures with high resolution.

TLC vs. HPLC

Type of Analysis

qualitative only

qualitative &

quantitative

Stationary Phase

2-dimensional

thin layer plate

3-dimensional

column

Instrumentation

minimal!

much! with many

adjustable parameters

Sample Application

spotting

(capillary)

injection

(Rheodyne injector)

Mobile Phase Movement

capillary action

(during development)

high pressure

(solvent delivery)

Visualization of Results

UV lightbox

on-line detection

(variable UV/Vis)

Form of Results

spots, Rfs

(retention factors)

peaks, Rts

(retention times)

Varian HPLC System9010 SolventDelivery System9050 VariableUV/Vis DetectorHPLC SolventReservoirsHPLCColumnRheodyneInjector9060 Polychrom(Diode Array) DetectorComputerWorkstationKeep an eye onthese 4 screens!

Varian Solvent Delivery System

Varian 9010 Solvent Delivery System Rheodyne Injector %A %B %C Flow Rate Pressure{H2O} {MeOH} (mL/min) (atmos.)

ReadyTernary PumpACBfrom solvent reservoirColumnto detectorto columnthroughpulsedampenerto injectorthrough pumploadinject

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

Varian 9060Polychrom DetectorUV SpectrumChromatogramResetReadyUV Spectrum{shows full UV abs.}Chromatogram{shows peaks, Rt}ABS.ABS.UVmaxUVmaxRtRt

HPLC ChromatogramsRt = 3.0 min.faster movingless retainedRt = 5.2 min.slower movingmore retained 0 1 2 3 4 5 6 7Time (minutes) Absorbance Approximationof peak area bytriangulationArea = base x height 2baseheightPeak APeak B

Chromatography Stationary Phasesrelatively polar surface relatively nonpolar surfaceSilica Gelnormal phasereversed phase

Normal vs. Reversed Phase Chromatography

Normal Phase

Reversed Phase

Stationary phase

Polar (silica gel)

Non-polar (C18)

Mobile phase

Non-polar

(organic solvents)

Polar

(aqueous/organic)

Sample movement

Non-polar fastest

Polar fastest

Separation based on

Different polarities

(functionality)

Different

hydrocarbon content