Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
Elution
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.
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
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:
• Increase in peak width and decrease in peak height (reduced sensitivity) for later
eluting peaks.
Elution
Isocratic vs. gradient elution
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.
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.
Elution
Isocratic vs. Gradient Elution