Introduction to HPLC

Basic Module in Bioorganic Chemistry January 16, 2007 Johannes Ranke

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Sources
R. Kellner, J.-M. Mermet, M. Otto, H. M. Widmer (Eds.), Analytical Chemistry, Wiley-VCH, Weinheim, 1998 W. J. Lough and I. W. Wainer (Eds.), High Performance Liquid Chromatography, Blackie Academic & Professional, London, 1996 Power Point slides P. Stepnowski/B. Jastorff W. M. A. Niessen, J. van der Greef, Liquid Chromatography Mass Spectrometry, Chromatography Series Volume 58, M. Dekker, New York, 1992 V. R. Meyer, Praxis der Hochleistungs- Flssigchromatographie, Salle + Sauerlnder, Frankfurt a. M., 1992

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1D Separation methods
2 1

1 2

Electrophoresis

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1D Separation methods
2 1 1 2

1 2

1 2

Electrophoresis

Chromatography

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1D Separation methods
2 1 1 2 1 2

1 2

1 2

1 2

Electrophoresis

Chromatography

Membrane separation

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1D Separation methods
2 1 1 2 1 2

1 2

1 2

1 2

Electrophoresis Separation according to Kinetic properties Equilibrium distribution Combinations thereof

Chromatography

Membrane separation

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Elution techniques
A+B

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Elution techniques

A B

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Elution techniques

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Elution techniques

Detector signal

4 Time [min]

10

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Equilibrium based separation

tM

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Equilibrium based separation

tM tR
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Equilibrium based separation

k =
nS nM

tM tR
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Equilibrium based separation

k =
nS nM

S t M t

tM tR
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Equilibrium based separation

k =
nS nM

S t M t

tR tM tM

tM tR
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Equilibrium based separation

k =
nS nM

S t M t

tR tM tM

k =

cS VS cM VM

tM tR
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Equilibrium based separation

k =
nS nM

S t M t

tR tM tM VS VM

k =

cS VS cM VM

=K

tM tR
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Peak shapes

c P. Stepnowski

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Column performance
S

wb = 4 tR t
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Column performance
S

N=

tR 2 ()

wb = 4 tR t
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Column performance
S

N=

tR 2 ()

tR 2 N = 16 ( w ) b

wb = 4 tR t
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Column performance
S

N=

tR 2 ()

tR 2 N = 16 ( w ) b

N = 8 ln 2

tR 2 ( w 0 .5 )

2 w0.5

wb = 4 tR t
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Column performance
N = 16
tR 2 ( wb )

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Column performance
tR 2 N = 16 ( wb ) t0 2 ) Ne = 16 ( tRw b

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Column performance
tR 2 N = 16 ( wb ) t0 2 ) Ne = 16 ( tRw b

H=

L N

H is the Height Equivalent of a Theoretical Plate (HETP).

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Column performance
tR 2 N = 16 ( wb ) t0 2 ) Ne = 16 ( tRw b

H=

L N

H is the Height Equivalent of a Theoretical Plate (HETP). tR,1 tR,2 Resolution RS = 0.5(wb,1 +wb,2 )

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The HPLC system

c P. Stepnowski

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Standard HPLC system

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Sample injection valve

c P. Stepnowski

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Detector properties
Sensitivity

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Detector properties
Sensitivity Selectivity

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Detector properties
Sensitivity Selectivity Linear range

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Detector properties
Sensitivity Selectivity Linear range Range of application

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Detector properties
Sensitivity Selectivity Linear range Range of application Analyte characterisation

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Detector properties
Sensitivity Selectivity Linear range Range of application Analyte characterisation Gradient elution?

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Detector properties
Sensitivity Selectivity Linear range Range of application Analyte characterisation Gradient elution? Relative cost

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UV/VIS detector

Lough/Wainer 1996, p. 121

S: light source W: wavelength selection BS: beam splitter M: mirror L: lens

C: ow cell RC: reference cell D: photodetector RD: reference photodetector

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UV/VIS detector

Meyer 1996, p.66

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UV cutoff wavelengths
Solvent Acetonitrile Water Cyclohexane Methanol Dichloromethane Cutoff wavelength (nm) 190 190 195 205 220

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The HPLC crash course

Preparation of the eluents

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The HPLC crash course

Preparation of the eluents Operating the device

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The HPLC crash course

Preparation of the eluents Operating the device Determination of dead time and dead volume

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The HPLC crash course

Preparation of the eluents Operating the device Determination of dead time and dead volume Separation of additional aromatic compounds

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The HPLC crash course

Preparation of the eluents Operating the device Determination of dead time and dead volume Separation of additional aromatic compounds Method development for two compounds

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The HPLC crash course

Preparation of the eluents Operating the device Determination of dead time and dead volume Separation of additional aromatic compounds Method development for two compounds

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In the lab
Start collection and evaluation of data during the experiment

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In the lab
Start collection and evaluation of data during the experiment Always collect information about chemicals

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In the lab
Start collection and evaluation of data during the experiment Always collect information about chemicals Use Prof. Jastorffs color coding scheme for discussing molecular interactions

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