organic chemistry i - lab high performance liquid chromatography hplc is characterized by the use of...
TRANSCRIPT
Organic Chemistry I - LabOrganic Chemistry I - Lab
High Performance Liquid Chromatography
HPLC is characterized by the use of high pressure to push amobile phase solution through a column of stationary phase
allowing separation of complex mixtures with high resolution.
TLC TLC vs.vs. HPLC HPLCType of Analysis qualitative only qualitative &
quantitative
Stationary Phase 2-dimensionalthin layer plate
3-dimensionalcolumn
Instrumentation minimal! much! with manyadjustable 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)
Varian HPLC SystemVarian HPLC System
9010 SolventDelivery System
9050 VariableUV/Vis Detector
HPLC SolventReservoirs
HPLCColumn
RheodyneInjector
9060 Polychrom(Diode Array) Detector
ComputerWorkstation
Keep an eye onthese 4 screens!
Varian Solvent Delivery SystemVarian Solvent Delivery System
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
Colum
n
to detector
to column
throughpulse
dampener
to injector
through pump
load
inject
Variable UV/Vis DetectorVariable UV/Vis Detector
ABS AUFS RunTime EndTime 0.001 2.000 238 0.00 min 10.0 min
Ready
Varian 9060Varian 9060Polychrom DetectorPolychrom Detector
UV Spectrum
ChromatogramReset
Ready
UV Spectrum{shows full UV abs.}
Chromatogram{shows peaks, Rt}
AB
S.
Time
AB
S.
Wavelength
UVmax UVmax
Rt Rt
HPLC ChromatogramsHPLC Chromatograms
Rt = 3.0 min.faster movingless retained
Rt = 5.2 min.slower movingmore retained
0 1 2 3 4 5 6 7
Time (minutes)
Abs
orba
nce
Approximationof peak area bytriangulation
Area = base x height
2
base
height
Peak A Peak B
Chromatography Stationary PhasesChromatography 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”
Normal vs. Reversed Phase ChromatographyNormal vs. Reversed Phase Chromatography
Normal Phase Reversed Phase
Stationary phase Polar (silica gel) Non-polar (C18)
Mobile phaseNon-polar
(organic solvents)Polar
(aqueous/organic)
Sample movement Non-polar fastest Polar fastest
Separation based onDifferent polarities
(functionality)Different
hydrocarbon content