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Lehrstuhl für Siedlungswasserwirtschaft

Ingenieurfakultät Bau Geo Umwelt


Technische Universität München

Normal and Reversed Phase Chromatography

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Normal and Reversed Phase Chromatography

• Adsorption Chromatography (Normal Phase and Reversed Phase)

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Normal and Reversed Phase Chromatography


Stationary Phase

Normal Phase Reversed Phase

Silica Si-OH Silica bonded phases


Silica bonded phases (-NH2; -CN) Octadecyl
Aluminiumoxide (Al-OH) Octyl
Phenyl
Cyanopropyl

Mobile Phase

Unpolar Solvents Polar Solvents

Heptane, Hexane, Dichlormethane, Buffer, methanol, acetonitrile


t-Butylmethylether, Cyclohexane,
Ethylacetate
PD Dr. J. Graßmann; PD Dr. T. Letzel
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Normal and Reversed Phase Chromatography


Normal Phase
The mostly used solid phases are silica gels (normal phase HPLC)

HO
OH O
Si
O Si OH O
O
O O Si
HO Si
O
HO OH Si O O
O O
Si O Si
Si
HO Si
O O
O

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Normal and Reversed Phase Chromatography


Normal Phase
The mostly used solid phases are silica gels (normal phase HPLC)

HO CH2 N
NH2 CH C
OH
(CH 2)n CH2 (CH 2)n
H 3C H3C H 3C
Si Si Si
O CH3 O CH3 O CH3
OH

Si Si Si Si
R R R R R R R R
R R R R
Silica Amino Diol Cyano

Column Retentivity: Silica >> Amino > Diol ~ Cyano


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

Normal and Reversed Phase Chromatography


Normal Phase
Normal Phase Advantages Normal Phase Disadvantages
 Separation selectivity can be greatly  Amendable to low and mid polarity
influenced by altering mobile phase samples
constituents and ratio of solvents  Controlling solvent strength can be
 Organic compounds are highly unpredictabal
soluble in the solvents  advantage  Solvents are more prone to air
for preparative chromatography bubble formation giving raise to
 Solvent viscosity is lower  higher instrument problems and noisy
flow rates and therewith higher baselines
sample thoughput can be achieved  Gradient elution is often not feasible
 Solvents used have higher cpst of
disposal and environmental impact

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Normal and Reversed Phase Chromatography


Reversed Phase
In case of modified silca gels, e.g. with alkyl groups: Reversed phase HPLC

HO H37 C18 O
OH O OC18 H37 O
Si Si
O Si OH O O Si OH O
O O
O O Si O O Si
HO Si H37 C18 O Si
O
HO OH Si O O
O H37 C18 O OC18 H37 Si O O
O O O
O Si Si O Si
Si O Si Si
H37 C18 O Si
HO Si O O
O O
O
O
PD Dr. J. Graßmann; PD Dr. T. Letzel
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Normal and Reversed Phase Chromatography


Reversed Phase

Example for transformation of a normal phase to a reversed phase:

Si OH Si OSi(CH 2)2R
Si OH Si OH
Si OH
Cl-Si(CH2)2R
Si OSi(CH 2)2R
Si OH Si OH
Si OH Si OH
Si OH Si OSi(CH 2)2R

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Normal and Reversed Phase Chromatography


Reversed Phase - Characteristics of different stationary phases
Si OSi(CH 2)2R
Si OH
H2)2R
Si OSi(CH 2)2R
Si OH
Si OH
Si OSi(CH 2)2R

R= Name
-(CH2)17-CH3 Octadecyl (C18) Decreasing
Hydrophobicity /
-(CH2)7-CH3 Octyl
Increasing Polarity
-CN Cyano
-(CH2)3-CN Cyanopropyl Increasing
-Phenyl Phenyl retention
of non-polar
-NH2 Amino analytes
-(CH2)3-NH2 Aminopropyl
PD Dr. J. Graßmann; PD Dr. T. Letzel
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Normal and Reversed Phase Chromatography


HILIC

HILIC = Hydrophilic Interaction Liquid – Chromatography

HILIC ia a variation of Normal Phase Liqiud Chromatography

The stationary phase is polar

The mobile phase has high organic content and is miscible with water

HILIC is suitable for separation of polar compounds

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Normal and Reversed Phase Chromatography


HILIC - Classification of HILIC stationary phases

Greco and Letzel. Sep. Sci. 2013, HILIC Solutions No.3


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

Normal and Reversed Phase Chromatography

Selection of a stationary phase


Types of Compounds Mode Stationary Phase Mobile Phase

Neutrals, Weak Acids, Reversed C18, C8, C4 Water/Organic


Weak Bases Phase cyano, amino Modifiers
Ionics, Bases, Acids Ion Pair C18,C8 Water/Organic
Ion-Pair Reagent
Compounds not soluble Normal Phase Silica, Amino, Organics
in water Cyano, Diol
Ionics / Inorganic Ions Ion Exchange Anion or Cation Aqueous/Buffer
Exchange Resin Counter Ion
High Molecular Weight Size Exclusion Polystyrene Gel Filtration -
Compounds Silica Aqueous
Polymers Gel Permeation-
Organic

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Normal and Reversed Phase Chromatography


Selection of stationary and mobile phase

Analyte examples (Log P gives the polarity)


OH
H3C CH3 H3C
OH O O HO
O
OH

Log P = -3.2 -1.8 -0.2 0.5 1.5 2.7 4.8

Analyte -4 -3 -2 -1 0 1 2 3 4 5
Log p
Normal Phase Separation Reversed Phase Separation
Column with hydrophilic surface Column with hydrophobic surface
Typical MeCN/H2O Hexane/EtOH H2O/MeCN
mobile (HILIC) CO2/MeOH H2O/MeOH
phases

PD Dr. J. Graßmann; PD Dr. T. Letzel