HPLC

1. 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
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, Rf’s
(retention factors)
peaks, Rt’s
(retention times)

3. Varian HPLC System
9010 Solvent
Delivery System
9050 Variable
UV/Vis Detector
HPLC Solvent
Reservoirs
HPLC
Colum
n
Rheodyn
e
Injector
9060 Polychrom
(Diode Array)
Detector
Computer
Workstation
Keep an eye on
these 4 screens!

4. Varian Solvent Delivery
System

5. Varian 9010 Solvent Delivery
System
Rheodyne
Injector
%A %B %C Flow Rate Pressure
{H2O} {MeOH} (mL/min) (atmos.)
Ready
Ternary Pump
A
C
B
from solvent
reservoir
Column
to
detector
to column
through
pulse
dampener
to injector
through pump
load
inject

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

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

8. HPLC Chromatograms
Rt = 3.0 min.
faster moving
less retained
Rt = 5.2 min.
slower moving
more retained
0 1 2 3 4 5 6 7
Time (minutes)
Absorbance

Approximation
of peak area by
triangulation
Area =
base x height
2
base
height
Peak A Peak B

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

10. 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