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
Type of Analysis Stationary Phase qualitative only 2-dimensional thin layer plate minimal!

vs.

HPLC
qualitative & quantitative

3-dimensional column Instrumentation much! with many adjustable parameters Sample Application spotting injection (capillary) (Rheodyne injector) Mobile Phase Movement capillary action high pressure (during development) (solvent delivery) Visualization of Results UV lightbox on-line detection (variable UV/Vis) Form of Results spots, Rfs peaks, Rts (retention factors) (retention times)

Varian HPLC System

9060 Polychrom (Diode Array) Detector Computer Workstation

HPLC Solvent Reservoirs

9010 Solvent Delivery System

9050 Variable UV/Vis Detector

Rheodyne Injector HPLC Column Keep an eye on these 4 screens!

Varian Solvent Delivery System

%A %B %C Flow Rate {H2O} {MeOH} (mL/min)

Pressure (atmos.)

to column
load

Ready

inject

Rheodyne Injector

Varian 9010 Solvent Delivery System

to injector

through pump

Column

through pulse dampener

C A B
from solvent reservoir

Ternary Pump

to detector

Variable UV/Vis Detector

ABS AUFS l RunTime EndTime 0.001 2.000 238 0.00 min 10.0 min

Ready

Ready

UV Spectrum
ABS.

UV Spectrum
{shows full UV abs.}
UVmax UVmax

Chromatogram
Reset

Wavelength

ABS.

Rt

Rt

Time

Chromatogram
{shows peaks, Rt}

Varian 9060 Polychrom Detector

HPLC Chromatograms
Absorbance

Approximation of peak area by

triangulation

Peak A

Peak B height

Time (minutes)
Rt = 3.0 min. faster moving less retained Rt = 5.2 min. slower moving more retained

base
Area = base x height 2

Chromatography Stationary Phases

Silica Gel
O O O | | | OSiOSiOSiOH | | | O O O | | | OSiOSiOSiOH | | | O O O

Derivatized Silica Gel

O O O | | | OSiOSiOSiOR | | | O O O | | | OSiOSiOSiOR | | | O O O

Where R = C18H37 hydrocarbon chain (octadecylsilyl deriv. silica or C18)

bulk (SiO2)x

surface

bulk (SiO2)x

surface

relatively polar surface normal phase

relatively nonpolar surface reversed phase

Normal vs. Reversed Phase Chromatography

Normal Phase Stationary phase Mobile phase Sample movement Separation based on Polar (silica gel) Non-polar (organic solvents) Non-polar fastest Different polarities (functionality) Reversed Phase Non-polar (C18) Polar (aqueous/organic) Polar fastest Different hydrocarbon content