Download Techniques of chromatography

Techniques of chromatography
Part 2
Techniques of chromatography
A.
B.
C.
D.
E.
F.
G.
H.
OPEN-COLUMN CHROMATOGRAPHY
PAPER CHROMATOGRAPHY (PC)
THIN-LAYER CHROMATOGRAPHY (TLC)
Gas chromatography
HIGH-PRESSURE LIQUID CHROMATOGRAPHY
SUPERCRITICAL FLUID CHROMATOGRAPHY (SFC)
ELECTROPHORESIS (Electrochromatography)
High Performance Capillary Electrophoresis (HPCE):
A. OPEN-COLUMN CHROMATOGRAPHY
stationary phase (silica or alumina) is packed in glass tubes
The stationary phase particle size is large (250 jam) to allow the
passage of solvent.
Disadvantage is the long time needed for the separation of
complex mixtures (up to one week or more).
B) PAPER CHROMATOGRAPHY (PC)
The adsorbent is a sheet of paper of suitable texture and thickness
Development may be ascending in which case the solvent is carried up the
paper by capillary forces, or descending, in which case the solvent flow is
also assisted by gravitational force.
C- Thin-layer chromatography
Separation is based on migration of the sample spotted on a coated (stationary phase) plate
with one edge dipped in a mixture of solvents (mobile phase).
However, it is not usually as accurate or sensitive as liquid chromatography.
D- Gas chromatography
Principle:
 A pressured gas flows through heated tube coated with
liquid stationery phase or packed stationery on a solid
support.
 The analyte loaded on the head of the column via heated
injection port, where it is evaporated.
The separation of a mixture occurs according the relative
time spent in the stationary phase
Instrumentation
1. Injection of the simples manually or using autosampler
usually size of 0.5 – 2 ul injection volume
2. The sample is evaporated and condensed at the head of the
column
3. The column either capillary or packed column, the mobile
phase is a gas to carry the sample through the column which
is Helium or nitrogen gases.
4. The oven to heat the column up to 400 oC.
5. The detector usually flame ionization detector FID
The Gas Chromatograph
WWU -- Chemistry
Gas Chromatography
Injection site
Control
panel
Capillary column
Gas Chromatography
Plotter
Gas Chromatography: Separation of a Mixture
WWU -- Chemistry
Stationery phase for GC
Types of columns
1. Packed columns:
Usually glass columns silanised to remove Si….OH
The column mobile phase used is nitrogen at flow rate of 20 ml/min.
Limitation,
can not be used above 280 oC because of the evaporation of the stationary
phase
2. Capillary column
The inner surface is coated with orange silicon polymers which are chemically
bonded to silanol groups
The mobile phase used usually Helium at low flow of 0.5 to 2 ml/min
Columns
• Packed
•
Capillary
Factors governing the retention of
compounds in capillary GC;
1. Carrier gas type and flow: Nitrogen and helium
2. Column temperature: increase of column temperature, decreases
resolution between two compounds,
3. Column length: increase the column length increases the resolution
4. Film thickness phase loading: the greater the volume of the stationary
phase the more solutes will be retained
5. The column internal diameter: the smaller the diameter the more
efficient
Gas Chromatogram
Highest
b.p.
Lowest
b.p.
Retention
time
Chromatograms - 551.1
Detectors
1. Flame ionization detector FID
Compounds burned at the detector produced ions
Detects carbon – hydrogen compounds till 10 ng
Wide application range up to 10-6
2. Electron capture detector ECD
Highly halogenated compounds can be detected at 50 fg – 1 pg
Wide application for drugs determination in biological fluids. Have wide application in
environmental analysis such as chlorofluorocarbons in the air
3. Nitrogen phosphate collectors
Used for compounds containing nitrogen and phosphors such as drugs and metabolities in body
tissues and fluids
High selective
4. Thermal conductivity detectors TCD
Responding to cooling effect of the analyte passing over filament
Insensitive, used for determination of water vapour such as in peptides
Application of GC
1. Detection of impurities in drug formulation
2. used for quantification of drug substances in
formulation specially for drugs lack of
chromophore
3. characterization of some row material used for
drug synthesis
4. measurements of drugs and their metabolites in
biological fluids
Limitation of GC
1. only thermostable compounds can be
analysed
2. the sample may require derivatisation to be
volatile
3. quantitative sample introduction is more
difficult due to the small volume of sample
injected
Derivatization: GC
 The technique is extended by the preparation of volatile derivatives of the
nonvolatile compounds or of the compounds, which undergo
decomposition.
 used also for improvement of peak shape, relocation of an interfering
peak, improvement of sensitivity or improvement of separation of closely
related compounds.
 An example of derivatization is silylation by addition of trimethylsilyl group
to carboxylic acids, amines, imines, alcohols, phenols and thiols by
treatment with hexamethyldisilazane.
Injector
Column
HPLC
Data Processing
Detector
Chromatographic Column
25
The system consists of main parts:
1Mobile phase or solvent reservoir.
2A high pressure pump.
3A sample inlet port.
4Column
5Detector
6Recorder
Schematic diagram of an HPLC unit
(1) Solvent reservoirs,
(2) Solvent degasser,
(3) Gradient valve,
(4) Mixing vessel for delivery of the mobile phase,
(5) High-pressure pump,
(6) Switching valve in "inject position",
(6') Switching valve in "load position",
(7) Sample injection loop,
(8) Pre-column (guard column),
(9) Analytical column,
(10) Detector (i.e. IR, UV),
(11) Data acquisition,
(12) Waste or fraction collector.
The pump, capable of maintaining high pressures draws the solvent
(mobile liquid phase) from the reservoir and pushes it through the column.
The sample is injected through a port into the high pressure liquid
carrier steam between the pump and the column.
The separation takes place on the columns, which vary, from 25-100 cm
length and 2-5 mm in internal diameter. Typical flow rates are 1-2 ml/min
with pressures up to several thousand psi.
The column effluent passes through a non-destructive detector where a
property such as :
UV absorbance,
Rl or
molecular fluorescence
To increase the efficiency of separation, the mobile phase may be altered
by changing its polarity, pH or ionic strength. HPLC offers the advantages of
speed, resolution and sensitivity.
There are two types of HPLC procedures:
1. LLC: the column consists of an inert support usually silica gel on which the
stationary partitioning phase is adsorbed.
 In the normal phase mode, the stationary phase is polar (e.g. methanol,
acetonitrile or water) while the mobile phase is less polar (e.g. iso-octane,
chloroform or n-hexane). This mode is usually used for the separation of
polar components.
 In the reverse phase LLC, the stationary phase is less polar and the mobile
phase is polar. It is usually used for the separation of non-polar components.
2. LSC: The packing may be silica (polar packing) or octadecylsilica, ODS (C18-silica,
non-polar packing). Adsorption mechanism is involved here.
 In the normal phase LSC, the packing is polar (silica) and the mobile phase is
less polar (e.g. n-hexane).
 In the reverse phase LSC, the packing is non-polar (eg. ODS) and the mobile
phase is polar (e.g. acetonitrile-water or methanol-water).
Again, as under LLC, normal phase LSC is used for polar solutes while reverse
phase LSC is used for separation of non-polar compounds.
Elution Approaches
• Isocratic - constant mobile phase composition
• Gradient - variable mobile phase composition
– step - change accomplished sharply at a defined
point in time
– continuous - change accomplished gradually over
time
HPLC Columns
• Analytical columns
Made of stainless stele
Internal diameter 2.1 – 4.6 mm
column long 30 – 300 mm
Particle size 3 – 10 micrometer
Gourd columns
Shorter column 7.5 mm Used to
prevent the adsorption of
substances on the analytical column
Stationary phase in HPLC
Chemically inert
Non-soluble in any imaginable mobile phase
Thermal and chemical stability
Appropriate physical sorption of analyte
Shape: Uniform spherical particles
Stationary phase in HPLC
1. unmodified silica stationary phases
Spherical and regular Particle size 3-10 uM;
Polar surface due to the silanol groups
Uses
For the separation and retention of non polar and moderately polar
compounds such as poly aromatics fats, oil, isomers
2. Modified silica stationary phases
Spherical and regular Particle size 3-10 uM:
Non-polar due to the modification of silanol groups by chloroaloxy silane
produces stable stationary phases, Ex ODS Octadecylsilane the most used
stationary phase in reversed phase chromatography
Uses
For the separation and retention of wider range of polar and moderately
polar substances such as drugs and amino acids
Si OH
+
CH3
Cl Si R
CH3
Si OH
CH3
Si O Si R
CH3
-HCl
Si O
-2HCl
+
SiR2
Cl2SiR2
Si OH
Si O
amino
octadecyl
NH2
CN cyano
O
Polar phase
octyl
Nonpolar phase
OH
OH
phenyl
diol
F) SUPERCRITICAL FLUID CHROMATOGRAPHY (SFC)
SFC , is a column chromatographic technique in which a supercritical fluid is
used as a mobile phase .The used mobile phase is frequently cooled to be
maintained in a liquid state for easier pumping to the column. Carbon dioxide is
the most frequently used mobile phase .Other mobile phases include ammonia,
nitrous oxide , and xenon .SFC ,is an intermediate between GC and HPLC and
offers the advantages of both .
A supercritical fluid: is a substance above its critical temperature and pressure.
Critical temperature (Tc): is that above which it is impossible to liquefy a gas, no
matter how great a pressure is applied.
Critical pressure (Pc): is the minimum pressure necessary to bring about liquefaction
at Tc.
Critical volume (Vc): is the volume occupied by one mole of gas or liquid at the critical
temperature and pressure.
Compound
Tc, °C
Pc, atm.
Carbon dioxide
31.05
72.9
Nitrous oxide
36.4
71.5
Ammonia
132.4
111.3
2-Propanol
235.1
47.6
Methanol
239.4
79.9
Acetonitrile
274.8
47.0
Water
374.1
217.6
Advantage of supercritical fluids as mobile phases in
chromatography compared with liquid chromatography is that
1. solutes generally have much higher diffusion coefficient in
them than in liquids. This leads to enhanced speed of
separation and possibly greater resolution with complex
mixtures, especially for large molecules.
2. SFC possesses also advantages over GC in that solutes do
not have to be volatile or thermally stable.
C) ELECTROPHORESIS (Electrochromatography)
Introduction:
Electrophoresis is a technique in which solutes are separated by their different rates
of travel through an electric field.
- commonly used in biological analysis, particularly in the separations of
proteins, peptides and nucleic acids
The rate of migration (electrophoretic mobility) of each species is a function of its
charge, shape and size.
Gel electropherograms
High Performance Capillary Electrophoresis (HPCE):
– another type of zone electrophoresis
– It involves high voltage electrophoresis in narrow bore fused-silica capillary tubes and
on-line detectors similar to those used in HPLC.
- On passing through the detector, they produce response profiles that are sharper than
chromatographic peaks.