Types of liquid chromatography

1 2

We focus on the stationary phase

chemistry:
• Normal and reversed phase
– Ion-pair chromatography
• Size exclusion chromatography
• Chiral chromatography
• Ion chromatography

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Reversed phase chromatography

Normal and reversed phase
chromatography
• Most common type of chromatography
– Main topic for the rest of the semester
• Stationary phases: -C18 and –C8 mostly
• Mobil phase: Water + MeOH/MeCN/THF
– Additives/buffer solution

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1
Reversed phase chromatography Analyted do not show any Normal phase chromatography
Analytes are very strongly bond retention.
to the stationary phase and do Analyte bonds very strongly to
not elute stationary phase and elutes very
slowly

Analyte
All compounds are eluted very
Eluent elutes only very
polar compounds from the
Analyte
fast. No separation. column
Eluent interacts with stationary phase too
strongly and therefore analytes elute too fast.

Eluent
Eluent

Statsionary phase
Non-polar Polar

Stationary phase
7 Non-polar Polar 8

Mobile phase: MeCN/water + 0.1% formic acid

Mobile phase – MeCN/water
5
OH
3 4
2 HO OH
OH
HO OH
OH
CH3
6 OH OH
7 8 OH CH3
CH3
9 10 N
OH
O O OH
CH3 CH3 H3C OH
N
O O

Hydrophobicity is important!
9 10

Mobile phase
• Mixture of organic solvents
Normal phase chromatography – Does not contain water

Stationary phase is more polar than

mobile phase

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2
 Stationary phase Analytes
• Anorganic adsorbent • Neutral substances
– Silica gel • Problems occur with ionic substances
– Aluminium oxide – Mobile phase additives
• Polar bonded phases
– Cyano
– Diol
– Amino

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Retention Stationary phase: silica gel

CH3 Eluent: hexane

• Is described as adsorption process N

O CH3

CH3 O O

– Stationary phase is covered with solvent

molecules O
N
CH3 O
N
O O
– Analyte retention occurs due to displacement of
solvent molecules

15 16

Problem
• Very polar compounds
– In normal phase chromatography bond too
strongly with the stationary phase and elute
HILIC very slowly from the column
– In reversed phase chromatography no retention
occurs and different analytes can not be
Hydrophilic interaction separated
chromatography
• If compound is ionic ion-chromatography
can be used

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3
 HILIC chromatography:
• Same stationary phases as in normal phase Example
chromatography
• Same eluents (MeCN/water) as in reversed
phase chromatography
• Mechanism is explained via liquid-liquid
partitioning
• Stationary phase is covered with water layer,
while the mobile phase contains less water
• Analyte partitions between the water layer and
the mobile phase
• Polar compounds show higher retention and
elute later 19 20

Introduction
• In Size Exclusion Chromatography (SEC)
Size Exclusion Chromatography two modes are used:
– Gel Filtration uses water-based eluent. Mostly
(SEC) used for protein separation.
– Gel Permeation Chromatography uses and
organic solvent. Used for synthetic polymer
separation.

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SEC principles of separation Column

• Depending on their size, molecules can

enter the pores of stationary phase.
– 1. Very large molecules can’t fit into the pores
and elute early.
– 2. Small molecules can enter and move within
the pores freely.
– 3. Middle sized molecules can enter the pores
partially. The smaller the molecule the longer it
takes to elute.
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4
 SEC properties SEC obtained information
• Separation is based on the hydrodynamic • Retention time average molecular weight
volume of the compounds (size). • Peak shape molecular weight distribution
• Allows to separate based on:
– Chain length
• Calibration is done with the same type of
– Location of functional groups
polymer as being analysed
– Functional groups
• Sample and calibration mixture are
– Geometrical structure
dissolved in the same solvent.
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Example of calibration curve

Example of chromatogram Larger molecules do not fit into the pores and
Analytes and their average molecular weights:
cannot be separated
670 kDa
240 kDa
69 kDa
MW
18.5 kDa
analysable
17 kDa
with this
253 Da column

Smaller molecules fit into all pores and

cannot be separated

27 28

Stationary phase is prepared

• Gel Filtration
– Dextran (linear glucose polymer) Ion (Exchange) Chromatography
– polyacrylamide
– Agarose
• Gel Permeation
– Extensively cross-linked copolymer of
polystyrene and divinylbenzene

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5
 Terminology IEC Retention
• Both terms IEC – ion exchange chromatography • Stationary phase is covered with charged groups:
and IC – ion chromatography are used – amine, quaternary ammonium – positively charged
– Sometimes as synonyms – sulfonate, carboxylate – negatively charged
• IEC covers the processes that occur on the surface • Retention is based on the following equilibria:
of ion exchange resin – R-K+ + X+  R-X+ + K+ (cation exchange)
• IC is the chromatography of ion separation and – R+Cl- + X-  R+X- + Cl- (anion exchange)
detection.
– Mostly conductivity cell is used as a detector. • Increasing the concentration of counterion (i.e. K+
– Separation is usually carried out with IE, but RP can and Cl-) decreases the analyte retention
also be used.
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1. Is influenced by eluent pH and analyte pKa

2. Is influenced by counterion concentration and properties
3. Is influenced by eluent pH and Stationary phase properties
Why IC?
• Why not use Reversed Phase Chromatography
(Ion-Pair Chromatography)?
• Detection
1 – Conventional detectors are blind to most inorganic (and
RNH3+  RNH2 + H+ some organic) compounds – conductivity cell
– For MS-detection eluent additives should be volatile,
2 3 ion-pair reagents (i.e laurylesulfate) usually are not
O O O
- +  -  • Preparative Chromatography
O Na O HO
– Non-volatile ionpair-reagents are problematic
Stationary phase • Suitable as the first step in multidimensional
chromatography
33 34

Acids occur in different forms depending on the eluent pH

α Eluent pH
increases
stationary phase
Interaction with

Fully deprotonated A-
• Analytes are usually acids or bases
• Ion Exchange is most effective when stationary
phase and analyte are charged oppositely
• Derive retention dependency
– For weak and strong cation/anion exchanger
Fully protonated HA – For strong/weak acid/base

pKa pH
Why acids/bases can not always be determined with reversed or
normal phase chromatography?
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6
 Different stationary phases are of different capacity
Weak and Strong ion exchange e.g. sulfonic acids R – SO2OH

resins
e.g. quaternary amines R-NR3+
• Cation exchange
– WCX and SCX – weak/strong cation exchange
• Anion exchange
– WAX ja SAX– weak/strong anion exchange
• Strong are applicable 2<pH<12 e.g. carboxylic acids R – COOH

• Weak resins lose charge at some pH

• Weak are seldom used
e.g. amines R-NR2H+
– To modify selectivity
– To decrease selectivity

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pH effects Salt effect on retention

• Silica based columns can not be used pH>8 • Retention depends on the anion (cation)
– Counter ions have different strength of displaceing the
and pH<1. analyte
• SCX and SAX with OH- may catalyze some • Strong counterions decreases analyte retention
reactions more then weak counterions with the same
concentration
– e.g. ester hydrolyses
– F- (weak)<OH-<CH3COO-<Cl-<SCN-<Br-<CrO4-<NO3-
<I-<SO42-(strong)
– Li+(weak)<H+<Na+<NH4+<K+<Rb+<Cs+<Mg2+<Ca2+<
Ba2+ (strong)

39 40

Organic solvent as an additive Method development

• Organic solvent decreases retention
• MeOH and MeCN can be used to alter
selectivity
• Column
– SAX for acidic and anionic compounds; SCX for basic
and cationic compunds;
• Eluent
– Start with water based buffer solution.
– pH>6 for SAX and pH<6 for SCX.
– If analyte pKa is known pH>pKa for anion exchange
and pH<pKa for cation exchange should be used
– concentration 20...50 mM

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7
 Ion exchange resins
Method development • Synthetic organic polymers
Most often used resins. Usually copolymers of
• Eluent component B styrene and divinylbenzene containing
– Buffer + salt (e.g. K2SO4) appropriate functional groups.
– Test gradient 0...100% B The biggest advantage is that synthetic polymers
• If analyte does not elute can be used in a wide pH range (0-13). Therefore
– Increase temperature also weak acids/bases can be determined.
– Add MeOH
– Use weak ion exchange resin
The biggest disadvantages is softness – high
• If retention is ok (0.5<k<20)
pressure can nt be used. This also limits column
– Adjust selectivity with modifying salt, pH and organic
lenght and flow rate.
additive.
43 44

Ion exchange resins Ion-exchange

Ion-exchange can be characterized:
• Synthetic organic polymers
Crosslinked polystyrene polymer: • Selectivity
– Stationary phase
– Analytes charge
– Solvated ions volume
– Analytes polarizability
– Ion-exchanger capacity
• Oxides – Functional groups on the stationary phase

45 46

Apparatus of ion-exchange Suppressor-column

chromatography
• Eluent high conductivity hinders usage of
conductivity detector
• Suppressor-column helps to remove ions:
High capacity anioon
+ - -
Low capacity exchange R OH +Cl
Alkali R+Cl-+H2O
cation-exchange
HCl metals
Conductivity
Buffer (10-3) Sample Column Supressor cell

• Needs frequent regeneration

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8
 Membran suppressor
Common applications
• Determination of inorganic ions
– Mostly anions
• Amino acids, peptides and proteins
• Nucleic acids

49 50

k
CH3

H3C
O Si
RCOO-
H3C
CH3
H3C
O
H3C Si CH3

H3C OH3CCH3 H+
Si
H3C

Ion-pair chromatography CH3 RCOOH

pKa
pH

Therefore: Retention of carboxylic acids depends on the eluent pH.

Same stands for weak bases.

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Mobile phase Mobile phase

• Ion-pair reagent • Optimization
– Opposite charge to the analyte – pH
– Long hydrophilic change – Concentration of ion-pair reagent
• Bonds with reversed phase
• In case of gradient both components have to
• Alkyl solfonates
contain ion-pair reagent with the same
– For cation determination
concentration.
• Tetraalkylammonium salts
– For anion determination

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9
 CH3
k Concentration of ion-pair reagent on the stationary phase depends on:
H3C
H3C CH3
RCOO- 1. ion-pair reagent concentration in eluent
O Si N
+
RCOO- 2. properties of ion-pai reagent
H3C
CH3 H3C CH3
H3C
O
H3C Si CH3
H3C CH3
H3C OH3CCH3
Si N
+ H+ O -
O
H3C H3C S
H3C CH3 Ion-pair reagnts
concentration in O

H3C CH3
CH3
RCOOH RCOOH the stationary
phase
+
N pKa

H3C CH3 pH O -
O
TBA H3C S
O

Surface is covered with positive charge!

Ion-pair reagnts
concentration in
the mobile phase

55 56

OH

CH3 O O
- BH+ H
N

H3C S analyte
O Si O
H3C
CH3 H3C HO
H3C
O
H3C Si CH3 O - OH
O
H3C OH3CCH 3
H3C
S H
Si O H3C N
- O
H3C S O Ion-pair reagent in eluent

O
CH3 HO

kanalyte A lot of ion-pai reagent in the OH

mobile phase that also compeat H

N
for analytes charge.

HO

H
Very littel ion-pair N

reagent bond to stationary

phase. Analyte retention
is weak. NH2

COncnetration of
ion-pair reagent 57 58
in the eluent

Enantiomer
• Stereoisomers, which are
– mirror images
Chiral separation – non-superimposable
• Same physical and chemical properties, except for
the direction in which they rotate the plane of
polarized light
• Nomenclature
– R and S
– D and L
– + and -
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10
 Diastereo(iso)mer Chiral separation
• Stereoisomers, which are not • Separation has to be carried out on chiral
– non-superimposable system:
– Are not mirror images – Chiral component in mobile phase
• Different physical and chemical properties – Chiral liquid stationary phase (liquid-liquid
partition chromatography)
– Chiral solid stationary phase
– After derivatization with chiral reagent

61 62

Chiral separation Chiral solid stationary phase

• Separation is based on the diastereomeric • Chiral compound is bond to stationary phase
complex between analyte and (CSP – chiral stationary phase)
– No one stationary phase is capable of separating all
chromatographic system. possible isomers
• Different types
– Brush-type CSP
– Helix-shaped phases
– Cavity phases
– Proteins
– Ligand-exchange phases
63 64

Stationary phase
Brush-type CSP H
O
H
H H
H
H N
N This oxygen can nto give
H
• Most common are Pirkle type CSP H R H hydroge bond with stationary
phase
H
– Designed according to 3-poind rule H H
H H H
– Work in both normal and reversed phase H O H H H H
H H
chromatography O H
H
N HH H N
HO H

N H H
– Is available in both isomeres – analytes elution O
O O
O
N
order is reversible H
H O
H
NO2 N
O O H
N
H2 O
S-isomer
H H H
C N C C N C O

R-isomer
O O

NO2
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