2 - Reversed Phase - Chromatography - 2

Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt

Technische Universität München
Reversed Phase Chromatography
PD Dr. J. Graßmann; PD Dr. T. Letzel

• The name „Reversed phase“ is due to the fact, that ist was
(chronologically) the second mode of chromatography after „Normal phase”
chromatography
• In Reversed phase chromatography the mobile phase is more polar than
the stationary phase.


Typical reversed phases are hydrophobic and chemically bonded to the surface
of a silica support particle. An example is the modification with C18 groups:
HO H37C18O
OH O OC18H37 O
Si Si
O Si OH O Si OH O
O O
O
O O Si O O Si
HO Si H37C18O Si
O
HO OH Si O O
O H37C18O OC18H37 Si O O
O O O
O Si Si O Si
Si O Si Si
HO H37C18O Si
Si O
O O O
O O


Typical stationary phases are modified with carbon chains of different length (C2
– C30) or with phenyl-, diol- or cyano groups:
HO CH2 N
CH3 CH C
OH
(CH 2)n (H2C) 3 CH2 (CH 2)n
H 3C H3C H3C H 3C
Si Si Si Si
O CH3 O CH3 O CH3 O CH3
Si Si Si Si
R R R R R R R R
R R R R
n = 1: C2 Cyano
3: C4 Phenyl Diol
7: C8
17: C18
29: C30
Depending on conditions Diol- and Cyanophases may also work in normal phase mode.

Mobile Phases in Reversed Phase Chromatography
… usually consist of water or buffer and an organic modifier.
Polarity Index Elution strength (for C18)

O
Water 10.2 - H H
Acetonitrile 5.8 3.1 H3C C N
O
Methanol 5.1 1.0 H3C H
H 2C CH2
Tetrahydrofuran 4.0 3.7 H 2C CH2
O


Separation in Reversed Phase Chromatography
The retention mainly is based on the hydrophobicity of an analyte molecule
HO
CH
3
The hydrophobic portion of
CH3
CH3
CH3 the analyte (example:
octanol) interacts with the
H3 C
unpolar chain (example: C8)
CH3
Si
CH
O 3
Si H 3C Si CH3
H 3C Si CH3 O
H 3C Si CH3
O Si
O
Si
Si
stationary phase


Separation in Reversed Phase Chromatography
HO
CH
3
The hydrophobic portion of
CH3
CH3
CH3 an analyte molecule has
large effect on separation,
H3 C
for example, butanol,
Si
O
CH
3
CH3 hexanol, and octanol can be
Si H 3C Si CH3 well separated on reversed
H 3C Si CH3 O
H 3C Si
O
CH3
O Si phase but not on normal
Si
Si
phase
stationary phase


Retention in Reversed Phase Chromatography
The hydrophobicity of an analyte can be expressed as Log P which is a

measure of an analytes partition between two immiscible solvents (usually
octanol and water)
The higher the Log P, the more hydrophobic is the molecule
The retention decreases in the following order:
Aliphatics > induced dipoles (e.g. CCl4) > permanent dipoles (e.g. CHCl3) > weak
Lewis bases (esters, aldehydes, ketones) > strong Lewis bases (amines) > weak
Lewis acids (alcohols, phenols) > strong Lewis acids (carboxylic acids)


Retention in Reversed Phase Chromatography
Retention order:
1. Straight chain hydrocarbon – most hydrophobic (C11)
2. Straight chain hydrocarbon – less hydrophobic (C10)
3. Branched n-hydrocarbon (C10)
4. Unsaturated C10 - (more polar due to π electron dipole in the
double bond)
5-7. Analytes with functional groups. Ionized analytes will elute
fastest of all


Influence of mobile phase strength on retention
Analytes have increasing Log P, i.e. increasing hydrophobicity
O
H3C O
Solvent A: Water
Acetophenone CH3
Solvent B: Acetonitrile
Isocratic at 25% Acetonitrile
Propiophenone
O
O O
Benzophenone O
CH3 H3C
H3C
Heptanophenone Octanophenone
Valerophenone
Retention time
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.

O O
H3C
Acetophenone
CH3
Solvent A: Water
Propiophenone Isocratic at 50% Acetonitrile
 shorter analysis time compared to 25% Acetonitrile
Benzophenone O
CH3 O
H3C O
H3C
Valerophenone Heptanophenone
Octanophenone
Retention time [min]


O
H3C Solvent A: Water
Acetophenone
O
Solvent B: MeOH
CH3
Isocratic at 50% MeOH
 longer analysis time compared to 50% Acetonitrile
Propiophenone  Acetonitrile is the stronger solvent
O
O
Benzophenone O
CH3
H3C O
H3C
Valerophenone Heptanophenone
Octanophenone


Influence of temperature on retention
Solvent A: Water
Temperature 20°C
O
O
O NH
H 3C CH3
Acetanilide
2-Acetylfuran


Influence of temperature on retention
Solvent A: Water
O
Temperature 50°C
 Shorter retention times
O
H 3C
O
2-Acetylfuran  Change in selectivity
NH
Acetanilide CH3


• In reversed phase chromatography the mobile phase is more polar than the
stationary phase
• Mobile phases in Reversed Phase Chromatography usually consist of water
or buffer and an organic modifier
• The retention mainly is based on the hydrophobicity (which can be expressed
as Log P) of an analyte molecule
• Retention is – amongst others - influenced by
• the solvent strength of the organic modifier
• the temperature (which may also influence selectivity)

2 - Reversed Phase - Chromatography - 2

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Lehrstuhl für Siedlungswasserwirtschaft

Ingenieurfakultät Bau Geo Umwelt

Reversed Phase Chromatography

PD Dr. J. Graßmann; PD Dr. T. Letzel

Reversed Phase Chromatography

PD Dr. J. Graßmann; PD Dr. T. Letzel

Reversed Phase Chromatography

PD Dr. J. Graßmann; PD Dr. T. Letzel

Reversed Phase Chromatography

Reversed Phase Chromatography

… usually consist of water or buffer and an organic modifier.

Polarity Index Elution strength (for C18)

Acetonitrile 5.8 3.1 H3C C N

PD Dr. J. Graßmann; PD Dr. T. Letzel

Reversed Phase Chromatography

The retention mainly is based on the hydrophobicity of an analyte molecule

PD Dr. J. Graßmann; PD Dr. T. Letzel

Reversed Phase Chromatography

The retention mainly is based on the hydrophobicity of an analyte molecule

PD Dr. J. Graßmann; PD Dr. T. Letzel

Reversed Phase Chromatography

The retention mainly is based on the hydrophobicity of an analyte molecule

The hydrophobicity of an analyte can be expressed as Log P which is a

The higher the Log P, the more hydrophobic is the molecule

The retention decreases in the following order:

PD Dr. J. Graßmann; PD Dr. T. Letzel

Reversed Phase Chromatography

PD Dr. J. Graßmann; PD Dr. T. Letzel

Reversed Phase Chromatography

Analytes have increasing Log P, i.e. increasing hydrophobicity

Reversed Phase Chromatography

Retention time [min]

Reversed Phase Chromatography

Retention time [min]

Reversed Phase Chromatography

Retention time [min]

Reversed Phase Chromatography

Retention time [min]

Reversed Phase Chromatography

PD Dr. J. Graßmann; PD Dr. T. Letzel

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