Introduction to HPLCBasic Module in Bioorganic Chemistry

January 16, 2007

Johannes Ranke

Introduction to HPLC – p.1/17

Sources

R. Kellner, J.-M. Mermet, M. Otto, H. M. Widmer (Eds.), AnalyticalChemistry, Wiley-VCH, Weinheim, 1998

W. J. Lough and I. W. Wainer (Eds.), High Performance LiquidChromatography, 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- Flüssigchromatographie,Salle + Sauerländer, Frankfurt a. M., 1992

Introduction to HPLC – p.2/17

1D Separation methods

1

1

2

2

Electrophoresis

Introduction to HPLC – p.3/17

1D Separation methods

1

1

2

2

Electrophoresis

1

1

2

2

Chromatography

Introduction to HPLC – p.3/17

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

1D Separation methods

1

1

2

2

Electrophoresis

1

1

2

2

Chromatography

1

1

2

2

Membrane separation

Separation according to

Kinetic properties

Equilibrium distribution

Combinations thereof

Introduction to HPLC – p.3/17

Elution techniques

A + B

Introduction to HPLC – p.4/17

Elution techniques

A

B

Introduction to HPLC – p.4/17

Elution techniques

A

B

Introduction to HPLC – p.4/17

Elution techniques

Time [min]

Det

ecto

r si

gnal

0 2 4 6 8 10

A

B

Introduction to HPLC – p.4/17

Equilibrium based separation

tM

Introduction to HPLC – p.5/17

Equilibrium based separation

tMtR

Introduction to HPLC – p.5/17

Equilibrium based separation

tMtR

k′ =nS

nM

Introduction to HPLC – p.5/17

Equilibrium based separation

tMtR

k′ =nS

nM=t̄S

t̄M

Introduction to HPLC – p.5/17

Equilibrium based separation

tMtR

k′ =nS

nM=t̄S

t̄M=tR−tM

tM

Introduction to HPLC – p.5/17

Equilibrium based separation

tMtR

k′ =nS

nM=t̄S

t̄M=tR−tM

tM

k′ =cS·VS

cM·VM

Introduction to HPLC – p.5/17

Equilibrium based separation

tMtR

k′ =nS

nM=t̄S

t̄M=tR−tM

tM

k′ =cS·VS

cM·VM= K ·

VS

VM

Introduction to HPLC – p.5/17

Peak shapes

c©P. Stepnowski

Introduction to HPLC – p.6/17

Column performance

t

S

tR

b b

2σ

b b

wb = 4σ

Introduction to HPLC – p.7/17

Column performance

t

S

tR

b b

2σ

b b

wb = 4σ

N = ( tRσ)2

Introduction to HPLC – p.7/17

Column performance

t

S

tR

b b

2σ

b b

wb = 4σ

N = ( tRσ)2

N = 16 · ( tRwb)2

Introduction to HPLC – p.7/17

Column performance

t

S

tR

b b

2σ

b b

wb = 4σ

N = ( tRσ)2

N = 16 · ( tRwb)2

N = 8 · ln 2 · ( tRw0.5)2

b b

w0.5

Introduction to HPLC – p.7/17

Column performance

N = 16 · ( tRwb)2

Introduction to HPLC – p.8/17

Column performance

N = 16 · ( tRwb)2

Neff = 16 · (tR−t0

wb)2

Introduction to HPLC – p.8/17

Column performance

N = 16 · ( tRwb)2

Neff = 16 · (tR−t0

wb)2

H =L

N

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

Introduction to HPLC – p.8/17

Column performance

N = 16 · ( tRwb)2

Neff = 16 · (tR−t0

wb)2

H =L

N

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

tR,1−tR,2

0.5·(wb,1+wb,2)

Introduction to HPLC – p.8/17

The HPLC system

c©P. Stepnowski

Introduction to HPLC – p.9/17

Standard HPLC system

Introduction to HPLC – p.10/17

Sample injection valve

c©P. Stepnowski

Introduction to HPLC – p.11/17

Detector properties

Sensitivity

Introduction to HPLC – p.12/17

Detector properties

Sensitivity

Selectivity

Introduction to HPLC – p.12/17

Detector properties

Sensitivity

Selectivity

Linear range

Introduction to HPLC – p.12/17

Detector properties

Sensitivity

Selectivity

Linear range

Range of application

Introduction to HPLC – p.12/17

Detector properties

Sensitivity

Selectivity

Linear range

Range of application

Analyte characterisation

Introduction to HPLC – p.12/17

Detector properties

Sensitivity

Selectivity

Linear range

Range of application

Analyte characterisation

Gradient elution?

Introduction to HPLC – p.12/17

Detector properties

Sensitivity

Selectivity

Linear range

Range of application

Analyte characterisation

Gradient elution?

Relative cost

Introduction to HPLC – p.12/17

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

Introduction to HPLC – p.13/17

UV/VIS detector

Meyer 1996, p.66

Introduction to HPLC – p.14/17

UV cutoff wavelengths

Solvent Cutoff wavelength (nm)

Acetonitrile 190Water 190Cyclohexane 195Methanol 205Dichloromethane 220

Introduction to HPLC – p.15/17

The HPLC crash course

Preparation of the eluents

Introduction to HPLC – p.16/17

The HPLC crash course

Preparation of the eluents

Operating the device

Introduction to HPLC – p.16/17

The HPLC crash course

Preparation of the eluents

Operating the device

Determination of dead time and dead volume

Introduction to HPLC – p.16/17

The HPLC crash course

Preparation of the eluents

Operating the device

Determination of dead time and dead volume

Separation of additional aromatic compounds

Introduction to HPLC – p.16/17

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

Introduction to HPLC – p.16/17

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

Introduction to HPLC – p.16/17

In the lab

Start collection and evaluation of data during theexperiment

Introduction to HPLC – p.17/17

In the lab

Start collection and evaluation of data during theexperiment

Always collect information about chemicals

Introduction to HPLC – p.17/17

In the lab

Start collection and evaluation of data during theexperiment

Always collect information about chemicals

Use Prof. Jastorff’s color coding scheme fordiscussing molecular interactions

Introduction to HPLC – p.17/17