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Lehrstuhl für Siedlungswasserwirtschaft

Ingenieurfakultät Bau Geo Umwelt


Technische Universität München

Normal Phase Chromatography

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Normal Phase Chromatography

• Normal phase chromatography was the first Liquid


Chromatographic technique Chlorophyll a

• In 1906 Mikhail Tsvet invented chromatography Chlorophyll b

• He used calcium carbonate as stationary phase and petrol


Lutein
ether/ethanol mixtures as mobile phase to separate plant
Violaxanthin
pigments
Neoxanthin
• In normal-phase chromatography the stationary phase is
more polar than the mobile phase.

"Chromatography of chlorophyll results". Licensed under CC BY-SA 2.5 via Wikimedia Commons -
http://commons.wikimedia.org/wiki/File:Chromatography_of_chlorophyll_results.jpg#mediaviewer/File:Chromatography_of_chlorophyll_results.jpg

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Normal Phase Chromatography


The mostly used solid phases are (modified) silica gels

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

Typical mobile phases are hexane, dichloromethane, t-butylmethylether or


ethylacetate

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Normal Phase Chromatography


The silica gel may be modified by amino-, diol- or cyano groups

HO CH2 N
NH2 CH C
OH
(CH 2)3 (CH 2)3 (CH 2)3
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

Depending on conditions Diol- and Cyanophases may also work in reversed phase mode.
PD Dr. J. Graßmann; PD Dr. T. Letzel
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Normal Phase Chromatography


Mobile Phases used in Normal Phase Chromatography

Solvent Elution strength Viscosity Refraction UV cut Boiling


for normal phase index off [nm] point [°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

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Normal Phase Chromatography


Separation in Normal Phase Chromatography

The retention mechanism is based on polar adsorption of either the mobile


phase molecule or the analyte onto the stationary phase

HC Analyte (example: butanol)

H3
3

adsorbs at stationary phase

C
The hydrocarbon portion of
the analyte is only weakly
attracted, whereas the polar
OH

hydroxyl functional group is


OH OH
strongly attracted.
Si

stationary phase

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Normal Phase Chromatography


Separation in Normal Phase Chromatography

HC
The hydrophobic portion of

H3
3

C
an analyte molecule has
little effect on separation,
for example, butanol,
hexanol, and octanol
OH

OH
OH cannot be well separated
Si using normal phase
chromatography (but they
stationary phase can be easily separated
using reversed-phase).

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Normal Phase Chromatography


Separation in Normal Phase Chromatography

The adsorption decreases in the following order:

carboxylic acids – amides – amines – alcohols – ketones – aldehydes - esters,


nitro compounds – aromatics - olefins - saturated hydrocarbons

The Elution order is reverse

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Normal Phase Chromatography


Separation in Normal Phase Chromatography

Retention is based on an adsorption process, i.e. interaction of analyte with the


surface of the column packing

Surface sites are fixed  location and spacing affects separation

 Normal Phase Chromatography can be used to separate isomers

Br
OH OH
R Si Br Br OH R Si Br OH
OH OH OH OH
R Si R
R Si
R R R
Si Si Si Si
R R R R
R R R R R R R R
R R R R
stationary phase stationary phase

m-Dibromobenzene interacts with p-Dibromobenzene interacts with only


two adjacent silanol groups one silanol group  retention is weaker
PD Dr. J. Graßmann; PD Dr. T. Letzel
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Normal Phase Chromatography


Separation in Normal Phase Chromatography

One further advantage is the possibility to separate very hydrophibic molecules

Those are strongly retained in reversed phase chromatography

But they are soluble in the unpolar solvents used in normal phase chromatography

An example ist the separation of fat soluble vitamins:

Picture from: Sigma-Aldrich, Merck (www.sigmaaldrich.com)

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Normal Phase Chromatography

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 soluble in unpredictabal
the solvents  advantage for ➢ Solvents are more prone to air bubble
preparative chromatography formation giving raise to instrument
➢ Solvent viscosity is lower  higher flow problems and noisy baselines
rates and therewith higher sample ➢ Gradient elution is often not feasible
thoughput can be achieved ➢ Solvents used have higher cost of
disposal and environmental impact

PD Dr. J. Graßmann; PD Dr. T. Letzel