hplc: an introduction
DESCRIPTION
High performance Liquid ChromatographyTRANSCRIPT
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Introduction to HPLCBasic Module in Bioorganic Chemistry
January 16, 2007Johannes Ranke
Introduction to HPLC p.1/17
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SourcesR. Kellner, J.-M. Mermet, M. Otto, H. M. Widmer (Eds.), AnalyticalChemistry, Wiley-VCH, Weinheim, 1998W. J. Lough and I. W. Wainer (Eds.), High Performance LiquidChromatography, Blackie Academic & Professional, London, 1996Power Point slides P. Stepnowski/B. JastorffW. M. A. Niessen, J. van der Greef, Liquid Chromatography Mass Spectrometry, Chromatography Series Volume 58, M.Dekker, New York, 1992V. R. Meyer, Praxis der Hochleistungs- Flssigchromatographie,Salle + Sauerlnder, Frankfurt a. M., 1992
Introduction to HPLC p.2/17
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1D Separation methods
1
1
2
2
Electrophoresis
Introduction to HPLC p.3/17
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1D Separation methods
1
1
2
2
Electrophoresis
1
1
2
2
Chromatography
Introduction to HPLC p.3/17
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1D Separation methods
1
1
2
2
Electrophoresis
1
1
2
2
Chromatography
1
1
2
2
Membrane separation
Introduction to HPLC p.3/17
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1D Separation methods
1
1
2
2
Electrophoresis
1
1
2
2
Chromatography
1
1
2
2
Membrane separationSeparation according to
Kinetic propertiesEquilibrium distributionCombinations thereof
Introduction to HPLC p.3/17
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Elution techniques
A + B
Introduction to HPLC p.4/17
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Elution techniques
A
B
Introduction to HPLC p.4/17
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Elution techniques
A
B
Introduction to HPLC p.4/17
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Elution techniques
Time [min]
D
e
t
e
c
t
o
r
s
i
g
n
a
l
0 2 4 6 8 10
A
B
Introduction to HPLC p.4/17
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Equilibrium based separation
tM
Introduction to HPLC p.5/17
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Equilibrium based separation
tMtR
Introduction to HPLC p.5/17
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Equilibrium based separation
tMtR
k =nS
nM
Introduction to HPLC p.5/17
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Equilibrium based separation
tMtR
k =nS
nM=tS
tM
Introduction to HPLC p.5/17
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Equilibrium based separation
tMtR
k =nS
nM=tS
tM=tRtM
tM
Introduction to HPLC p.5/17
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Equilibrium based separation
tMtR
k =nS
nM=tS
tM=tRtM
tM
k =cSVS
cMVM
Introduction to HPLC p.5/17
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Equilibrium based separation
tMtR
k =nS
nM=tS
tM=tRtM
tM
k =cSVS
cMVM= K
VS
VM
Introduction to HPLC p.5/17
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Peak shapes
cP. Stepnowski
Introduction to HPLC p.6/17
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Column performance
t
S
tR
b b
2
b b
wb = 4
Introduction to HPLC p.7/17
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Column performance
t
S
tR
b b
2
b b
wb = 4
N = ( tR)2
Introduction to HPLC p.7/17
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Column performance
t
S
tR
b b
2
b b
wb = 4
N = ( tR)2
N = 16 ( tRwb)2
Introduction to HPLC p.7/17
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Column performance
t
S
tR
b b
2
b b
wb = 4
N = ( tR)2
N = 16 ( tRwb)2
N = 8 ln 2 ( tRw0.5)2
b b
w0.5
Introduction to HPLC p.7/17
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Column performance
N = 16 ( tRwb)2
Introduction to HPLC p.8/17
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Column performance
N = 16 ( tRwb)2
Neff = 16 (tRt0
wb)2
Introduction to HPLC p.8/17
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Column performance
N = 16 ( tRwb)2
Neff = 16 (tRt0
wb)2
H =L
N
H is the Height Equivalent of a Theoretical Plate (HETP).
Introduction to HPLC p.8/17
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Column performance
N = 16 ( tRwb)2
Neff = 16 (tRt0
wb)2
H =L
N
H is the Height Equivalent of a Theoretical Plate (HETP).Resolution RS = tR,1tR,20.5(wb,1+wb,2)
Introduction to HPLC p.8/17
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The HPLC system
cP. Stepnowski
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Standard HPLC system
Introduction to HPLC p.10/17
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Sample injection valve
cP. Stepnowski
Introduction to HPLC p.11/17
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Detector properties
Sensitivity
Introduction to HPLC p.12/17
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Detector properties
SensitivitySelectivity
Introduction to HPLC p.12/17
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Detector properties
SensitivitySelectivityLinear range
Introduction to HPLC p.12/17
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Detector properties
SensitivitySelectivityLinear rangeRange of application
Introduction to HPLC p.12/17
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Detector properties
SensitivitySelectivityLinear rangeRange of applicationAnalyte characterisation
Introduction to HPLC p.12/17
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Detector properties
SensitivitySelectivityLinear rangeRange of applicationAnalyte characterisationGradient elution?
Introduction to HPLC p.12/17
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Detector properties
SensitivitySelectivityLinear rangeRange of applicationAnalyte characterisationGradient elution?Relative cost
Introduction to HPLC p.12/17
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UV/VIS detector
Lough/Wainer 1996, p. 121
S: light source C: flow cellW: wavelength selection RC: reference cellBS: beam splitter D: photodetectorM: mirror RD: reference photodetectorL: lens
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UV/VIS detector
Meyer 1996, p.66
Introduction to HPLC p.14/17
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UV cutoff wavelengths
Solvent Cutoff wavelength (nm)Acetonitrile 190Water 190Cyclohexane 195Methanol 205Dichloromethane 220
Introduction to HPLC p.15/17
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The HPLC crash course
Preparation of the eluents
Introduction to HPLC p.16/17
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The HPLC crash course
Preparation of the eluentsOperating the device
Introduction to HPLC p.16/17
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The HPLC crash course
Preparation of the eluentsOperating the deviceDetermination of dead time and dead volume
Introduction to HPLC p.16/17
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The HPLC crash course
Preparation of the eluentsOperating the deviceDetermination of dead time and dead volumeSeparation of additional aromatic compounds
Introduction to HPLC p.16/17
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The HPLC crash course
Preparation of the eluentsOperating the deviceDetermination of dead time and dead volumeSeparation of additional aromatic compoundsMethod development for two compounds
Introduction to HPLC p.16/17
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The HPLC crash course
Preparation of the eluentsOperating the deviceDetermination of dead time and dead volumeSeparation of additional aromatic compoundsMethod development for two compounds
Introduction to HPLC p.16/17
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In the lab
Start collection and evaluation of data during theexperiment
Introduction to HPLC p.17/17
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In the lab
Start collection and evaluation of data during theexperimentAlways collect information about chemicals
Introduction to HPLC p.17/17
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In the lab
Start collection and evaluation of data during theexperimentAlways collect information about chemicalsUse Prof. Jastorffs color coding scheme fordiscussing molecular interactions
Introduction to HPLC p.17/17
Sources1D Separation methodsElution techniquesEquilibrium based separationPeak shapesColumn performanceColumn performanceThe HPLC systemStandard HPLC systemSample injection valveDetector propertiesUV/VIS detectorUV/VIS detectorUV cutoff wavelengthsThe HPLC crash courseIn the lab