Lehrstuhl für Siedlungswasserwirtschaft

Ingenieurfakultät Bau Geo Umwelt

Technische Universität München

Elution

PD Dr. J. Graßmann; PD Dr. T. Letzel

Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Elution

• Mobile Phase: A fluid which percolates through or along the stationary bed, in
a definite direction. It may be a liquid (Liquid Chromatography) or a gas (Gas
Chromatography) or a supercritical fluid (Supercritical-Fluid Chromatography).

• Isocratic Elution: The procedure in which the composition of the mobile phase
remains constant during the elution process.

• Gradient Elution: The procedure in which the composition of the mobile phase
is changed continuously or stepwise during the elution process.

PD Dr. J. Graßmann; PD Dr. T. Letzel

Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Elution
Mobile Phases
Some important characteristics of solvents
Solvent Elution strength for Viscosity Refraction UV cut off Boiling point
normal phase index [nm] [°C]
Fluoroalkanes -0.19 0.4 1.267 210 50
n-pentane 0 0.23 1.3575 195 36
n-hexane 0 0.33 1.3749 190 69
Chloroform 0.31 0.57 1.4457 245 61
Acetone 0.43 0.32 1.3587 330 56
Acetonitrile 0.50 0.37 1.3441 190 82
Isopropanol 0.6 2.3 1.3772 210 82
Ethanol 0.68 1.20 1.3614 210 78
Methanol 0.73 0.60 1.3284 205 65
Acetic acid High 1.26 1,3719 260 118
Water Higher 1.00 1.333 <190 100
Buffers Highest
PD Dr. J. Graßmann; PD Dr. T. Letzel
 Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Acetic acid
Acetone Elution
Acetonitrile
Benzene
Butyl Alcohol
Carbon Tetrachloride (CCl4)
Chloroform
Miscibility of solvents:
Cyclohexane
Cyclopentane
Miscible
Dichloroethene (C2H2Cl2) Not miscible
Dichloromethane (CHCl2)
Dimethylformamide (DMF)
Dimethylsulfoxide (DMSO)
Dioxan
Ethylacetate
Ethanol
Diethylether
Heptane
Hexane
Methanol
Methyethylketone (MEK)
Isooctane
Pentane
Isopropylalcohol
Dipropylether
Tetrachloroethane (C2H2Cl4)
Tetrahydrofuran (THF)
Toluene
Trichloroethene (C2HCl3)
Water
Xylene

Isopropylalcohol
Cyclopentane

Dipropylether
Cyclohexane

Ethylacetate

Diethylether
Acetonitrile

Chloroform
Acetic acid

Isooctane
Methanol
Benzene

Heptane

Pentane
Acetone

Toluene
C2H2Cl2

C2H2Cl4
Hexane
Butanol

Ethanol

C2HCl3
Dioxan

Xylene
DMSO
CHCl2

Water
MEK
DMF
CCl4

THF
PD Dr. J. Graßmann; PD Dr. T. Letzel
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Elution
Isocratic and Gradient Elution

100
Percentage organic solovent

Gradient: Final %B
Composition of mobile Purging
Phase changes over time Conditioning

Gradient time

Isocratic
Hold time Isocratic: Equilibration
Composition of mobile phase is constant

0
Initial % B Time
PD Dr. J. Graßmann; PD Dr. T. Letzel
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Elution
Isocratic Elution
Poorly resolved early eluting peaks

Composition of mobile phase is constant

Poor peak shape and poor

sensitivity for late eluting
compounds

Chromatogramms generated with HPLC Simulator (hplcsimulator.org); P.G. Boswell, D.R. Stoll, P.W. Carr, M.L. Nagel, M.F. Vitha, G.A. Mabbott, J. Chem. Educ. 90 (2013) 198-202.
PD Dr. J. Graßmann; PD Dr. T. Letzel
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Elution
Isocratic Elution
Problems with Isocratic Analysis:

• Poor resolution of early eluting peaks.

• Increase in peak width and decrease in peak height (reduced sensitivity) for later
eluting peaks.

• Long analysis times due to a wide sample polarity

• Column contamination by strongly retained components.

PD Dr. J. Graßmann; PD Dr. T. Letzel

Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Elution
Isocratic vs. gradient elution

Isocratic: 40% Acetonitrile

Gradient: 5-95% Acetonitrile in

25 minutes

Chromatogramms generated with HPLC Simulator (hplcsimulator.org); P.G. Boswell, D.R. Stoll, P.W. Carr, M.L. Nagel, M.F. Vitha, G.A. Mabbott, J. Chem. Educ. 90 (2013) 198-202.

PD Dr. J. Graßmann; PD Dr. T. Letzel

Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Elution

Effect of changing
gradient slope

Chromatogramms generated with HPLC Simulator (hplcsimulator.org); P.G. Boswell, D.R. Stoll, P.W. Carr, M.L. Nagel, M.F. Vitha, G.A. Mabbott, J. Chem. Educ. 90 (2013) 198-202.

PD Dr. J. Graßmann; PD Dr. T. Letzel

Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München
Elution
Isocratic vs. Gradient Elution
Advantages of Gradient Elution:
• Improved resolution
• Increased sensitivity of later eluting
analytes
• Ability to separate complex samples
• Improved peak shape
• Shorter analysis times — especially for
analytes whose polarity differ widely
• Reduced column deterioration due to
strongly retained components
PD Dr. J. Graßmann; PD Dr. T. Letzel
Disadvantages of Gradient Elution:
• More expensive instrumentation
• Possible precipitation at interfaces,
when using multiple proportioning
valves
• Re-equilibration time adds to analysis
time
• Instruments vary in their dwell volume
(VD), which can cause method transfer
problems