1

Separation Techniques
Separation principle
Solutes within a solution or volatiles within a
gas are placed in a mobile phase and passed
over a selected ~adsorbent material (the
stationary phase).
The solutes or volatiles have differential
~affinity for the absorbent material and
thus separate. For a given system, each
compound has a ~unique retention time.
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Separation Techniques
Distillation
Crystallization.
Solvent-partitioning.
Paper-chromatography.
Thin-layer chromatography.
Open column chromatography.
Separation Techniques
HPLC.
Normal-phase
Reverse phase
Size-exclusion chromatography.
Supercritical Fluid Chromatography.
Ion-chromatography.
Counter-current-chromatography.
Capillary-electrophoresis.
Gas-chromatography.
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Separation Techniques
GSC GLC
GAS SFC
NP RP EC
GPC GFC
SEC
Column
TLC Paper
Planar
LQUD
CHROMATOGRAPHY
Separation Techniques
Choice oI method
4
Separation Techniques
Choice oI method
MolSize-
Polarity-
Method
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Separation Techniques
Paper-chromatography
Principle
Partition chromatography. Analytes are separated based
on the interactions between two non-miscible liquid
phases according to their relative solubility. The main
diIIerence oI paper chromatography is that a sheet oI
paper is used Ior the inert phase.
Stationary phase: Water that is tightly bound to the
cellulose structure and Iill interspaces oI the paper Iibers.
Mobile phase: Any solvent (also known as a developing
solvent) that is partially miscible in water. Choice oI the
solvent will depend on the nature oI the substances to be
separated. OIten, a mixture oI two or three solvents is
required.
Separation Techniques
Paper-chromatography
Ascending Descending
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Separation Techniques
Paper-chromatography
2-Dimensional technique. Chromatography developed
twice in diIIerent directions with diIIerent mobile
phases.
ADSORPTION
CHROMATOGRAPHY
SurIace interactions
Dipole - Dipole (Normal Phase)
Intermolecular Iorces resulting Irom the attraction oI the positive
and negative ends oI the dipole moments oI polar molecules
Dipole - Induced Dipole (Normal Phase)
Even iI a molecule has no dipole moment a temporary dipole
moment can be induced when one molecule with a dipole
approaches another molecule in which the electrons are slightly
displaced Irom a symmetrical arrangement
Hydrophobic (Reversed Phase)
StereospeciIic (Chiral)
All interactions are competitive
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Separation Techniques
1hin Layer Chromatography
Plate (glass, aluminium, plastic) as support Ior
stationary phase (silica, RP-silica, modiIied with CN,
amino, phenyl, diol-Iunctions, cellulose, Al
2
O
3
.
Developing chamber; holds the mobile phase; single
solvent complex mixtures, and provides 'gas-
phase, which is important Ior separation process.
Thin Layer Chomatography
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Separation Techniques
1hin Layer Chromatography
Solvent is pulled through stationary phase by
capillary Iorces.
Variation 'rotating disc; Cyclo-graph.
Forced-Ilow (200bar pressure)
Separation Techniques
1hin Layer Chromatography
Also 2-D-Technique applicable.
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Separation Techniques
1hin Layer Chromatography
Detection of compounds
Spray reagents; color reactions Maillard reaction,
derivatization .
Iodine (dissolves in analytes).
Silica layer has a UV-absorbing (254nm)
compound.
UV-scanners.
FT-IR.
Raman.
MS
Radio-activity detection
Separation Techniques
HPLC
Parts of HPLC: pump, injector, column,
detector.
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Separation Techniques
HPLC
Injector
Separation Techniques
HPLC
Pump
typically constant flow pumps, no pulsations.
Pressure changes depending on viscosity of
solvent.
For packing constant pressure pumps.
Different options to do gradients:
High-pressure mixing Low pressure mixing
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InIluence oI solvent composition
A
B
C
D
E F
A
BC
DE
F
A
B
C
D
E
F
30 MeCN
70 20mM
Phosphate, pH6.95
50 MeCN
50 20mM
Phosphate, pH6.95
80 MeCN
20 20mM
Phosphate, pH6.95
A. Phenvlethvlamine
B. Pvridine
C. 2-Picoline
D. 2,4 Lutidine
E. 4-ethvlpvridine
F. 2,3 dimethvlaniline
Separation Techniques
HPLC
Gradient operation: we change solvent composition
over time.
RP: polar unpolar
Normal-phase: unpolar polar.
Speeds up separation.
Better separation.
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Separation Techniques
HPLC
Gradient operation:
gradient
isocratic
Separation Techniques
HPLC
Column.
Most commonly used 4.6mmx 250mm.
Many variations possible.
Packing material: SiO
2
, RP-8, RP-18, CN,
Phenyl..
DiIIerent particle sizes. The smaller the better,
however the larger the pressure. Typical 5m
packing.
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Reversed Phase
SEPARATION PRINCIPLE
Nonpolar (nonspeciIic) interactions oI analyte
with hydrophobic adsorbent surIace (-C18, C8,
Phenyl, C4)
DiIIerent sorption aIIinities between analytes
results in their separation
More polar analytes retained less
Analytes with larger hydrophobic part are retained longer
Almost no separation oI structural isomers
Reversed Phase
SEPARATION PRINCIPLE (2)
NonspeciIic (hydrophobic) interactions
are at least ten times weaker than polar
small diIIerences in component molecular
structure could have a signiIicant eIIect in their
retention
Reversed Phase Normal
Phase
Octane, k` 4.3 Octanol, k` 3.5
Nonane, k` 7.8 Nonanol, k` 3.3
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REVERSED-PHASE INTERACTIONS
nfluence of the eluent hydrophobicity
80 Octadecylsilica (ODS, C18)
10 Octylsilica (C8)
5 Butylsilica (C4)
3 Phenyl
2 Cyano (CN)
REVERSED PHASE MATERIAL
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Separation Techniques
HPLC
Column. EIIect oI packing
NORMAL PHASE
30 Silica Gel
47 Silica based bonded phases
Diol
Amine
Cyano
3 Alumina
20 Chiral Bonded Phases
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Normal Phase
ADSORPTION MECHANISM
Competitive polar interactions
The more polar component, the more stronger it
will be adsorbed and so retained longer.
Physical adsorption is a dynamic reversible
process.
Polarity oI the eluent plays major role in
separation
The more polar eluent, the more competitive it is
Ior adsorption sites and hence analyte will be
less retained.
Normal Phase:
SEPARATION PRINCIPLE
Polar (speciIic but nonionic) interactions
oI analyte with polar adsorption sites
(SiOH, -NH
2
, -CN, Diol) cause its
retention
DiIIerent sorption aIIinities between
analytes result in their separation
More polar analytes retained longer
Analytes with larger number oI polar Iunctional
group are retained longer
Structural isomers are oIten separated
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Normal Phase INTERACTIONS
nfluence of the eluent polarity
Separation Techniques
HPLC
Detectors:
UV.
Fluorescence.
MS.
(IR).
ReIractive index.
electrochemical (Ox/Red potential).
Conductivity.
Evaporative Light Scattering.
NMR.
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Separation Techniques
HPLC
Separation Techniques
HPLC Detectors
Refractive index:
Measures change in reIractive index oI
solvent. DiIIerential measurement.
Two types
DeIlection detector reIlective
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Separation Techniques
HPLC Detectors
Electrochemical cell:
Conductivity :
Amperometric cells
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Separation Techniques
HPLC Detectors
Evaporative Light Scattering:
Separation Techniques
Size-exclusion
Separation strictly by
size.
Biochemical
applications :
Proteins
Polymer
characterization
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Separation Techniques
Supercritical Fluid chromatography
Mobile phase
consists oI CO
2
.
Easy removal oI
solvent.
Sometimes diIIerent
separation.
Separation Techniques
SFC
ModiIiers :
1: isopropanol
2: acetonitrile
3: dichloromethane
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Separation Techniques
SFC
Comparison to other methods:
Separation Techniques
SFC
Gradient in SFC is
pressure.
Asymptotic gradient gives
better resolution.
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Separation Techniques
SFC
Again comparison to other methods (Gradient)
GC: temperature.
HPLC : solvent system/polarity.
SFC : pressure, temperature, solvent
system/polarity.
GC
LC
SFC
Separation Techniques
Ion-Chromatography
Determination oI Ions in solution.
Because oI the chromatographic technique
simultaneous determination oI several ions.
Concentration oI low concentrated samples,
separation Irom interIering matrix.
Detection limit ppb ( 1-5 g/L), depends on
detectors, Ions.
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Separation Techniques
Ion-Chromatography
Separation-process
Ion-Exchange
Ion exclusion
Ion-pairing
Chelation
Separation Techniques
Ion-Chromatography
Ion-Exchange
A charged analyte competes
in Ilowing solution with
eluent (mobile phase) oI a
like charge Ior sites having
the opposite charge
(stationary phase).
Key oI separation is
diIIerence in aIIinity.
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Separation Techniques
Ion-Chromatography
Ion-Exclusion
Stationary phase is ion-
exchange resin.
Analyte has same charge as
stationary phase.
An electric potential is
established between dilute
mobile phase, and high ion-
exchange concentration.
Separation Techniques
Ion-Chromatography
Ion-Exclusion
High ion-exchange concentration dictates high
counterion concentration to achieve electroneutral
conditions.
DiIIusion Iorces equalize counterion concentrations in
mobile, and stationary phase stationary phase
charged (DONNAN POTENTIAL).
Neutral species enter stationary phase, charged species
are repelled, or excluded (Ion-Exclusion)
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Separation Techniques
Ion-Chromatography
Ion-Pairing:
relies upon the addition of ionic compounds to the mobile
phase to promote the formation of ion pairs with charged
analytes. These reagents are comprised of an alkyl chain with
an ionizable terminus
When used with common hydrophobic HPLC phases in the
reversed-phase mode, ion pair reagents can be used to
selectively increase the retention of charged analytes
Separation Techniques
Ion-Chromatography
Examples:
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Separation Techniques
Capillary-electrophoresis
Ion migration through a capillary is achieved via
strong electric Iield.
Strong Iield induces electro osmotic Ilow.
A double layer develops on silica surIace.
Separation Techniques
Capillary-electrophoresis
Solvent migrates towards the cathode or anode.
Silica negatively charged, with buIIer cations Iorm 'double-
layer.
Cations oI solvent are pulled towards cathode (-), they drag the
solvent.
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Separation Techniques
Capillary-electrophoresis
Advantage: Flow proIile
Separation Techniques
Capillary-electrophoresis
Detection limits:
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Separation Techniques
Capillary-electrophoresis
Application: Anions, Cations, Biomolecules
Separation Techniques
Counter-Current-Chromatography
A separation technique that invoIves two Iiquid phases
fIowing in opposite direction.
AnaIogous to other chromatographic methods: The
stationary phase and the mobiIe phase resuIt in a
reIative countercurrent motion.
Both stationary and mobiIe phases are Iiquids.
Separation is based on the partition co-efficient ( ) of
the components in mixture.
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Separation Techniques
Counter-Current-Chromatography
For a given substance "A" the partition coefficient
is defined as:

A
= [A] upper phase/[A] lower phase

A
is a constant at any given temperature, and
unaffected by the presence of other substances
or concentration of the soIute in question.
A]
A]
Separation Techniques
Counter-Current-Chromatography
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Separation Techniques
Counter-Current-Chromatography
Separation Techniques
AIIinity-Chromatography
Stationary phase is modiIied with 'active
Biomolecule.
Two types oI ligands:
SpeciIic Antiody/Antigen
Group-speciIic
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Separation Techniques
AIIinity-Chromatography
Types oI ligands:
Enzymes
Antibody
Separation Techniques
AIIinity-Chromatography
Adsorption process.
Typically low Ilow to ensure binding.
loading washing elution
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Separation Techniques
Gas-Chromatography
Samples are evaporated
into gas-phase.
A stream oI inert gas
'carries the molecules
through the column.
Interaction with a
liquid stationary phase
separates out various
compounds.
Separation Techniques
Gas-Chromatography
Gradient mode in GC means temperature programmable oven.
DiIIerent types oI columns.
packed columns ; Iairly large, short columns; can be used in preparative
mode.
Capillary column; small diameter
DiIIerent detectors.
FID (Ilame ionization detector)
ECD (electron-capture detector)
AED (atomic emission detector)
TCD (thermal conductivity detector)
MS
IR
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Separation Techniques
Gas-Chromatography
Packed columns ;
Glass, metal, teIlon tubes
short columns; 2-3m length, 2-4 mm diameter.
packed with solid support
Soli support coated with 0.05 1 m oI liquid
Capillary columns:
Fused silica colums
SurIace modiIied through silanation.
No permanent absorption oI analytes (problem with Iree OH-
groups).
Separation Techniques
Gas-Chromatography
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Separation Techniques
Gas-Chromatography
FID: analyte is burned ions change in conductivity
FID is mass-sensitive (the more carbons the more ions!).
FID-signal also depends on Iunctional groups.
The 'STANDARD detector.
Separation Techniques
Gas-Chromatography
ECD
Electron gets emitted, causes ionization oI
gas. When organic compounds present a
constant current can be established.
TCD
Thermal conductivity oI helium 6-
10 times greater than organic
compoundsdramatic change in
presence oI analyte.temperature
oI Iilament changes.