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Lehrstuhl für Siedlungswasserwirtschaft

Ingenieurfakultät Bau Geo Umwelt


Technische Universität München

Ion Chromatography

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Ion Chromatography
• Ion chromatography can be subdivided into
o Ion exchange chromatography
o Ion exclusion chromatography
o Ion pair chromatography

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Ion Chromatography

Ion Exchange Chromatography

• Ion exchange chromatography can be used to


o Replace one ion by another
o Separate ions
o Remove all ions

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Ion Chromatography

Ion Exchange Chromatography

• An ion exchanger consists of an insoluble matrix to which charged groups


have been covalently bound. The matrix may be based on inorganic
compounds, synthetic resins or polysaccharides.
• The presence of charged groups is a fundamental property of an ion
exchanger.
• The type of group determines the type and strength of the ion exchanger;
their total number and availability determines the capacity.

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Ion Chromatography

insoluble matrix
charged groups

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Ion Chromatography

Ion Exchange Chromatography

3
3

CH
CH
Negatively charged Positively charged
analyte analyte

+
-

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Ion Chromatography
Ion Exchange Chromatography
Mg2+ Ca2+ Ca 2+ Ca2+ Mg2+ Hard water: high Ca2+ and Mg2+
2+ 2+
Mg2+ Ca Mg concentration

Na OOC
+-
Na OOC
+
Na

+-
-
O
-
+
Na+ - CO Resin with Na+ ions
O Na OOC
CO +- COO- Na+
Na OOC
Na+ -OOC COO- Na+
Application to soften Na+ -OOC
COO- Na+
COO- Na+

Na OOC
water: replacement

CO
OOC

+-

O
-
Na OOC
C

M
O

Na + -
OOC

of calcium and O-

g
+-

2+
O-
N
a+
Na + -

CO
magnesium by -
OOC
-
COO - Ca
2+

sodium -
OOC
2+ -
-
COO Ca
2+ COO The Ca2+ and Mg2+ ions replace
Mg OOC CO
O- the Na+ ions

OOC
CO Ca 2
CO O -Ca 2+
- +
Na + - O
OOC

O- -
OOC CO
Ca 2
Na + -

+-
OOC
COO- Mg2+
COO-
C
O
OOC

O-
N
a+
Na + -

Na+ Na+
Na+ Na+ Soft water: Ca2+ replaced by Na+
PD Dr. J. Graßmann; PD Dr. T. Letzel
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Ion Chromatography
Ion Exchange Chromatography

Application to soften water

..in household

Image: Rainer Knäpper, Free Art License

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Ion Chromatography
Ion Exchange Chromatography
Application to separate e.g. proteins

Polymer beads with


positively charged
groups

Large net negative charge

Net negative charge

Net positive charge


Large net positive charge

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Ion Chromatography
Ion Exchange Chromatography

Steps in an ion exchange separation


Negatively charged buffer ions

Column is Sample is applied Uncharged or equal Increasing the ionic strength of


equilibrated charged molecules elute the buffer displaces bound
with buffer during or just after molecules as ions from the
sample application buffer compete for binding sites
PD Dr. J. Graßmann; PD Dr. T. Letzel
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Ion Chromatography
Ion Exchange Chromatography
Application to remove all ions
SO3-H+ SO3-H+ CH2NR+3OH- CH2NR+3OH-

H3C CH2 CH CH2 HC CH2 CH CH2 CH2 CH3 H3C CH2 CH CH2 HC CH2 CH CH2 CH2 CH3

SO3-H+ CH2NR+3OH-

H3C HC CH2 CH CH2 CH CH2 CH2 CH2 CH3 H3C HC CH2 CH CH2 CH CH2 CH2 CH2 CH3
SO3-H+ SO3-H+ CH2NR+3OH- CH2NR+3OH-

H3C CH2 HC CH2 CH CH2 CH CH2 CH2 CH3 H3C CH2 HC CH2 CH CH2 CH CH2 CH2 CH3

Mixed bed containing negative and positive charged groups. The


respective counter ions are H+ and OH-, respectivley
PD Dr. J. Graßmann; PD Dr. T. Letzel
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Ion Chromatography
Ion Exchange Chromatography
Application to remove all ions
Ca2+ Cl- NO - Mg2+
Water containing Mg2+Mg2+ 3
SO42- Cl- SO42-
Cl Ca
2+
several ions -
Mg2+ Na+ Na+ - 2+ NO3-
Cl Ca
NO3- Cl- Na+ Ca2+
Cl-
Cl- Na Ca2+Cl
-
H+ NO3-
+ NO3-
Mixed bed: OH- - H+OH- OH- OH-
Cationic particles OH- OH H+
Ions bind to
OH- OH- + H+ OH OH-
-
NO3- Na+ SO42-
with OH- counter H Mg2+ OH- particles and
OH- OH- H+ OH- replace OH-
ions and anionic H+
H+ H+ Na+ SO42- Ca2+
particles with H+ H+ OH- H+ and H+
H+ OH- OH- H+ H+ Mg2+ Ca2+ Ca2+
counter ions H+ OH OH-
-
Mg2+ Mg2+ Cl- Cl
-
H+ H+ H+ H+
H+ H+
H+ OH-
H+ OH-
OH-H+ -H
+ OH- OH- H+ Deionized
OH OH- H+ water leaves
OH-
OH- H
+
OH- H+ the exchanger
H+ OH-
PD Dr. J. Graßmann; PD Dr. T. Letzel
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Ion Chromatography
Ion Exchange Chromatography

Application to remove all ions

..in the laboratory

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Ion Chromatography
Ion Exclusion Chromatography

Mainly used for separation of weak acids like carboxylic acids or amino
acids

The separation depends on the acid strength and on the pH of the mobile
phase

The strength of an acid is given by the pKa value

The smaller the pKa, the stronger the acid

At pH equal to the pKa, the concentration of protonated and deprotonated


species are equal

The separation is based on whether the acid is protonated (uncharged) or


deprotonated (charged)

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Ion Chromatography
Ion Exclusion Chromatography

Mainly used for separation of weak acids like carboxylic acids or amino acids

Due to the high density of


charged groups in the
stationary phase a potential
difference is generated which
leads to the formation of a
virtual membrane – the Donnan
membrane. This charged
membrane only permits neutral
molecules to reach the
stationary phase.
Charged molecules can not
pass the Donnan membrane

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Ion Chromatography
Retention in ion exclusion chromatography

The primary mechanism is the ion exclusion process which depends on the ratio of the
concentrations of ionized to neutral forms

pKa The completely dissociated (charged)


strong acids are eluted at the column
dead volume. Very weak acids are
eluted at the sum of the column dead
and inner volumes.

However, in separation of the acids


other mechanisms also play a role,
e.g. the length of the aliphatic chain or
double bounds etc.

Elution Volume
Tanaka, K. and Ishizuka, T., 1979: Elution Behavior of Acids in Ion-Exclusion Chromatography using a Cation Exchange resin, Journal of
Chromatography, 174 (1979) 153-157
PD Dr. J. Graßmann; PD Dr. T. Letzel
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Ion Chromatography
Retention in ion exclusion chromatography

Peak Nr. Acid pKa

OH O
HO
1 Tartaric 2.98 OH
O OH
O
4 Formic 3.7
H OH Increasing pKa

O
5 Acetic 4.8
H3C OH

O
8 Butyric 4.8 Same pKa
H3C OH
Increasing chain
O
9 Valeric 4.8 H3C
OH

http://www.dionex.com/en-us/products/columns/ic-rfic/ion-exclusion-packed/ionpac-ice-as1/lp-73268.html
PD Dr. J. Graßmann; PD Dr. T. Letzel
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Ion Chromatography
Ion Pair Chromatography

• Used to separate ionic analytes on a reversed phase column

• For substances which are charged too strongly for adsorption


chromatography and are not water soluble enough for ion
chromatography

• An organic salt containing a large organic counterion is added to the


mobile phase as an ion pairing reagent

• Typical ion pair reagents are: alkylsulphonates, alkylsulphates, tetra-


alkylammonium ions etc.

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Ion Chromatography
Ion Pair Chromatography
Typical Ion pairing reagents

Quarternary ammonium salts for anionic analytes

Alkylsulfonates for cationic analytes

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Ion Chromatography
Ion Pair Chromatography

Two mechanisms for separation are discussed:

• The analyte is paired in solution with the ion pair reagent and the neutral
complex undergoes interactions with the stationary phase

• The ion pair reagent populates the hydrophobic surface and the analyte
molecules undergo an ion exchange interaction

• Or…a combination of both

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Ion Chromatography
Ion Pair Chromatography
• The analyte is paired in solution with the ion pair reagent and the neutral
complex undergoes interactions with the stationary phase

analyte +-
ion pair reagent
 Neutral pair
 Interaction with stationary phase

-+
CH3
CH3

H 3C Si CH3
H 3C Si CH3
O
O
Si
Si

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

Ion Chromatography
Ion Pair Chromatography
• The ion pair reagent populates the hydrophobic surface and the analyte
molecules undergo an ion exchange interaction
Analyte undergoes ion
- exchange interaction

+
ion pair reagent populates stationary phase
 Negative charges on surface

-
-

CH3
CH3 CH3
CH3

H 3C Si CH3
H 3C Si CH3 H 3C Si CH3
H 3C Si CH3 O
O O
O Si
Si Si
Si

stationary phase stationary phase

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Ion Chromatography
Ion Pair Chromatography

Number of
Peak Nr. Compound
Carbon atoms

1 Methanesulfonic acid 1

2 1- Propanesulfonic acid 3

3 1- Butanesulfonic acid 4 Increased chain length


Increased hydrophobicity
4 1- Hexanesulfonic acid 6
Decreased charge ratio
5 1- Heptanesulfonic acid 7

6 1- Octanesulfonic acid 8

7 1- Decanesulfonic acid 10

http://www.dionex.com/en-us/products/columns/lc/reversed-phase/ionpac-ns1/lp-71767.html
PD Dr. J. Graßmann; PD Dr. T. Letzel
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Ion Chromatography
• Ion chromatography can be subdivided into
o Ion exchange chromatography
o Ion exclusion chromatography
o Ion pair chromatography

• Ion exchange chromatography can be used to


o Replace one ion by another
o Separate ions
o Remove all ions

• Ion exclusion chromatography is mainly used for separation of weak acids

• Ion pair chromatography is used to separate ionic analytes on a reversed


phase column

PD Dr. J. Graßmann; PD Dr. T. Letzel