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. (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

4. Varian Solvent Delivery System

5. 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

6. Variable UV/Vis Detector
ABS AUFS l RunTime EndTime
min min
Ready

7. 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

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

10. Normal vs. Reversed Phase Chromatography