Transcript HPLC Presentation - Drexel University

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
qualitative only
Stationary Phase
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, Rf’s
peaks, Rt’s
(retention factors)
(retention times)
Varian HPLC System
9060 Polychrom
(Diode Array) Detector
HPLC Solvent
Reservoirs
9010 Solvent
Delivery System
Computer
Workstation
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)
to column
Pressure
(atmos.)
load
Ready
inject
Rheodyne
Injector
Varian 9010 Solvent Delivery System
to injector
Column
through pump
C
through
pulse
dampener
A
B
Ternary Pump
from solvent
reservoir
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
UV Spectrum
{shows full UV abs.}
Chromatogram
Reset
UVmax
ABS.
UVmax
ABS.
Wavelength
Rt
Rt
Time
Chromatogram
{shows peaks, Rt}
Varian 9060
Polychrom Detector
Approximation
of peak area by
HPLC Chromatograms
Absorbance 
triangulation
Peak A
Peak B
height
0
1
2
3
4
5
6
Time (minutes)
Rt = 3.0 min.
faster moving
less retained
Rt = 5.2 min.
slower moving
more retained
7
base
Area =
base x height
2
Chromatography Stationary Phases
Silica Gel
Derivatized Silica Gel
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
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
bulk (SiO2)x
surface
relatively polar surface
relatively nonpolar surface
“normal phase”
“reversed phase”
Where R = C18H37
hydrocarbon chain
(octadecylsilyl deriv.
silica or “C18”)
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