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Modern Analytical
Chemistry
Chromatography
CHROMATOGRAPHY
Chromatography basically involves the
separation of mixtures due to differences in the
distribution coefficient (equilibrium distribution)
of sample components between 2
different
phases.
One of these phases is a mobile phase and
the other is a stationary phase.
OH
OH
OH
OH
Al
Al
Al
Al
Al
Acidic: -Al-OH
Neutral: -Al-OH + -Al-OBasic: -Al-O-
Si
O
O
Si
OH
OH
Si
O
O
Si
O
O
O
Si
O
O
Si
O
O
Si
OH
OH
O
O
Si
O
O
Si
O
O
Si
O
O
O
O
O
O
Si
O
O
S ample
(A+B+C)
Eluant (eluate)
Normal phase LS
Reverse phase LS
Si - O - H
Silica Gel
OH
HEXANE
Si - OH
CH3
OH CH
3
C-CH3
CH3
CH3- C
CH3
CH3
WATER-SOLUBLE VITAMINS
1.
Niacinamide
2.
Pyridoxine
H3C
N
HO
CONH 2
Riboflavin
CH 2OH
HOCH
HOCH
HOCH
CH 2
H 3C
N
N
CH 2OH
CH 2OH
3.
H 3C
4. Thiamin
O
NH
N
O
H3C
N
N
NH 2
CH 2
S
N
CH 2CH 2OH
Cl
CH 3
WATER-SOLUBLE VITAMINS
2
3
Inject
1
10
15
20
LIQUID-LIQUID CHROMATOGRAPHY
ODPN(oxydipropionylnitrile)
Normal Phase LLC
Reverse Phase LLC
NCCH3CH2OCH2CH2CN(Normal)
CH3(CH2)16CH3 (Reverse)
Chromatography Schematic
LIQUID
MOBILE PHASE
FORMAT
Liquid-Liquid
Chromatography
(Partition)
STATIONARY
PHASE
Normal Phase
Mobile Phase Nonpolar
Stationary phase Polar
Liquid
Reverse Phase
Mobile Phase Polar
Stationary phase Nonpolar
Liquid-Solid
Chromatography
(Adsorption)
Solid
Normal Phase
Reverse Phase
ION-EXCHANGE CHROMATOGRAPHY
SO3 Na
REMEMBER
MECHANISM OF ION-EXCHANGE
CHROMATOGRAPHY OF AMINO ACIDS
pH2
SO3
Na
H3N
COOH
Ion-exchange Resin
SO3
H3N
Na
+
-
COO
pH4.5
Mobile Phase
H3N
SO3 Na+
COOH
+
Na
SO 3
OH
H3 N
COOH
Exchange Resin
-
SO3 H3N+
COOH
SO 3
pH3.5
OH
H3 N
+
+
-
Na
COO
OH = H 2 O
Na
SO3
H3 N
+
-
COO
OH = H 2 O
SO 3Na+
pH4.5
GEL-PERMEATION CHROMATOGRAPHY
SOLVENTS
Polar Solvents
Water > Methanol > Acetonitrile > Ethanol >
Oxydipropionitrile
Non-polar Solvents
N-Decane > N-Hexane > N-Pentane >
Cyclohexane
LC Mode
Positional isomers
LSC or LLC
LSC or LLC
LSC or LLC
Ionizable Species
IEC
LLC
GCC
Detectors
1.
Ultraviolet Detector
200-400nm
254 nm
2.
HPLC Chromatography
1. Pump System. Mobile phase pressures up to 6000 psi are
Schematic of an HPLC
System
HPLC System
Pump System
Desirable Features:
Must generate pressures
up to 6,000 psi
HPLC Detectors
Absorbance Detectors
Diode
Array
Detecto
r
HPLC Detectors
HPLC Column
Typical dimensions
10-30 cm long
1-3 cm ID
Type of
HPLC
Depends
on:
1. Molecular
weight of
solute
2. Water solubility
of the solute
3. Polarity of the
solute
4. Ionic/non-ionic
character of
the solute
Separation Principles in
HPLC
General Rule of Thumb:
Polarity of analytes polarity of stationary
phase polarity of mobile phase
To achieve good separation, the analytes
should interact with the stationary phase,
but not too strongly or the retention time
will become very long
Reversed order
of elution
Increasing Mobil
phase Polarity,
Decreases
Elution Time
Typical Applications of
HPLC Chromatography
Field of Application Separation
Pharmaceuticals
Biochemical
Food Products
Clinical Medicine
Pollutants
2.
3.
4.
5.
6.
7.
8.
9.
10.
R
C
H 3 N+
COO-
Characterization
cell
Protein purification:
general experimental
setup
Column
chromatography
Homogenize
Centrifugation
Characterization
UV
time
XX X X
X
XX
X
XX
XX
X
X
- Met Ala Asn Cys His Glu Ser Thr Glu Arg-COOH
H2N
- Met Ala Asn Cys His Glu Ser Thr Glu Arg-COOH
H2N
His: 6.0
Glu: 4.1
Arg: 12.5
N-terminal amine: 8.0
C-terminal acid: 3.1
--
+ -
+-+
+
+-
-+
1.
2.
3.
4.
5.
-+
--+
-- - --
2.
3.
Reverse Phase
Column Chromatography
Reverse phase
Silica is chromatography
alkylated with long chain hydrocarbon groups, using 18
carbons long. This is usually referred to as C-18 silica.
Summary of Methodology
One of the main aims of biochemistry is to
characterize and catalogue all proteins in the cell
We have discussed some important tools for
separating proteins based on physical properties
such as size, affinity, charge.
Chromatography methods: ion exchange, affinity,
gel permeation chromatography
Electrophoresis: iso electric focusing, SDS PAGE,
2D gels (in the Biochemistry lecture series)
Overview of Paper
Chromatography
Works on principle of
Partition.
Important Concepts in
Paper Chromatography
Paper/TLC
Chromatography
Animation
0%
20%
50%
70%
Concentration of Isopropanol
100%
Works mainly on
principle of
adsorption.
Stationary phase is
the solid on the plate.
Thin-Layer Chromatography: A
Two-Component Mixture
solvent front
component B
Less polar!
component A
More polar!
solvent front
component B
component A
origin mixture
solvent front
origin
origin
Thin-Layer Chromatography:
Determination of Rf Values
solvent front
Rf of component A =
component B
dA
dS
dS
Rf of component B =
dB
dB
dS
The Rf value is a decimal
fraction, generally only
reported to two decimal
places
component A
dA
origin
Thin-Layer
Chromatography
: Qualitative
Analysis
B unknown
Visualization Method
Ultraviolet light
Iodine vapors to stain spots
Colored reagents to stain spots
Reagents that selectively stain spots while
leaving others unaffected.
TLC Advantages
Advantages over paper:
Its faster
It gives a better separation.
It is more versatile as the solid on the
plate can be varied.
Uses of TLC
Gas-Liquid Chromatography
Gas-Liquid Chromatography
Gas-Liquid Chromatography
Gas Liquid
Chromatography
Gas Chromatography
Filters/Traps
Data system
H
RESET
Regulators
Syringe/Sampler
Inlets
Detectors
Gas Carrier
Hydrogen
Air
Column
gas system
inlet
column
detector
data
system
Detector electronics
- 220 volts
Flame
Chassis ground
Jet
Column
Signal output
Gas-Liquid Chromatography
C18
C16
8
6
C14
4
2
0.5
1.0
1.5
2.0
2.5
Retention Time
The content % of C fatty acids =
14
C + C + C
3.0
Another GC Use:
TENTATIVE IDENTIFICATION OF UNKNOWN COMPOUNDS
Response
Mixture of known compounds
Octane
1.6 min = RT
Hexane
Response
1.6 min = RT
Decane
GLC ADVANTAGES
1.
2.
3.
4.
5.
Quantitatively analyzed
Fatty Acids
R C OH + CH3 OH + H2 SO4
Reflux
R C O CH3
Volatile in Gas
Chromatography
O
CH 2 O C R
O
CH
O C R
O
CH 2
O C R
CH 3 ONa
+
CH 3 OH
O
3 R C O CH3
Volatile in Gas
Chromatography
Stationary Phase
Paper
Trapped water
in the paper
Thin Layer
Oxide Coating
Column
Oxide packing
or resin
Gas-Liquid
Oxide or
volatile liquid
on a solid
support
High Performance
Liquid
Mobile Phase
amino acid
mixtures
food colors or
dyes
Organic Solvent
detect amino
acids
composition of
dyes and food
colors
Organic Solvent
Oxide Packing
or Resin or
Molecular Sieve
Typical Application
preparative
separation of
plant pigments
Organic Solvent
analysis of oil
mixtures
detect drugs &
steroids
fruit esters
Gas
analyze foods,
pesticides, etc
detect iron in
body fluids
Liquid