Advanced Analytical Chemistry 1
Separation Technique, Chromatography
For Postgraduate (Master's degrees)
Lecture No. 1
By
Asst. Prof. Dr. Layla Salih Al-Omran
Chromatography
Background
Chromatography is a technique in which substance are separated, purified and identified
from a mixture for qualitative and quantitative analysis. The sample is allowed to interact
or react with two immiscible phases: they are Mobile phase and Stationary phase
The stationary phase which may be a solid or a liquid supported on a solid, and the mobile
phase may be either liquid or gas.
the distribution of a molecule between the two phases is given by a distribution
coefficient, Kd =
How two molecules can be separated from each other?
When the components of the mixture have different physical and chemical properties which
can be used as a criterion to separate them
Physical Properties
Molecular weight, Boiling point (in case both are liquid), Freezing point, Crystallization
Solubility, Density.
Chemical Properties:
Functional Group, for example, phenol has –OH whereas aniline has NH2.
Reactivity towards another reagent to form complex
Example: Chemical Structure and physical Properties of benzene, phenol and aniline.
Classifications of chromatography
1. Based on the mechanism of separation:
Adsorption Chromatography
Partition Chromatography
Ion Exchange Chromatography
Size exclusion chromatography (gel filtration chromatography)
Affinity Chromatography
2. Based on the nature of the mobile phase:
1. Gas chromatography
• Gas liquid chromatography is partition chromatography
• Gas solid chromatography is adsorption chromatography
2. Liquid chromatography
Liquid chromatography can be further divided according to the mechanism of the separation.
For the liquid chromatography:
If a glass column is used, it
If the stationary phase is is column chromatography.
a thin layer then it is thin
layer chromatography.
If the stationary phase
is paper then it is paper
chromatography.
3. Supercritical fluid chromatography
Gas chromatography
Gas chromatography (GC) is a term used to describe the group of analytical
separation techniques used to analyze volatile substances in the gas phase.
The mobile phase is a chemically inert gas (such as helium, nitrogen etc.) that
carry the molecules of the analyte through the column.
There are tow types gas-solid chromatography(GSC) and gas- liquid
chromatography (GLC).
In gas-solid chromatography, solid adsorbent is used as a stationary phase &
separation is through adsorption process while in gas- liquid chromatography,
the stationary phase consists of thin layer of non-volatile liquid bound to solid
support & separation is through the process of partition.
Advantages of gas chromatography
High separation efficiency and analysis speed, a general sample analysis can be completed
in 20 minutes.
Small sample consumption and high detection sensitivity: 1 ml of gas sample consumption,
0.1 µl of liquid sample consumption.
Gas chromatography has good selectivity and can be used to analyse a mixtures and samples
with close boiling points. For example, some isotopes, cis-trans isomers,, optical
isomers, etc.
Wide range of applications, although mainly used to analyse gases and volatile organic
substances under certain conditions, it can also be used to analyse high boiling point
substances and solid samples.
Disadvantages of gas chromatography
It can only be used to analyse volatile substances. Some highly polar substances can be derived
to increase their volatility for GC analysis, but the process can be complex and may introduce
errors in quantitative analysis.
Instrumentation
all the chromatographs (GSC or GLC) consists of six basic components
1-carrier gas, 2-Sample injection system, 3-Separation column, 4-Oven or
Thermostat chambers, 5-Detectors, 6- Recorder system.
How does gas chromatography work? The sample is injected into the GC inlet (3)
through a septum which enables the
injection of the sample mixture without
losing the mobile phase.
Connected to the inlet is the analytical
column (4), a long (10 – 150 m), narrow
(0.1 – 0.53 mm internal diameter) fused
silica or metal tube which contains the
stationary phase coated on the inside walls.
The analytical column is held in the
column oven which is heated during the
analysis to elute the less volatile
components.
The outlet of the column is inserted into
the detector (5) which responds to the
chemical components eluting from the
column to produce a signal. The signal is
recorded by the acquisition software on a
computer to produce a chromatogram (6).
Instrumentation
1. Carrier Gas (mobile phase)
A carrier gas should be inert, dry & free of oxygen. Helium, Nitrogen, argon & hydrogen
gases are used as carrier gas
Carrier gas is supplied at high pressure & is passed to instrument at a rapid & reproducible
rate.
The table below shows advantages and disadvantages of He and N2 carrier gases.
Sample injection system
A sample port is necessary for introducing
the sample at the head of the column.
A calibrated microsyringe is used to transfer
a volume of sample through a rubber septum
and thus into the vaporization chamber.
Most of the separations require only a small
fraction of the initial sample volume and a
sample splitter is used to direct excess
sample to waste.
Commercial gas chromatographs involve the use of both split and splitless
injections when alternating between packed columns and capillary columns.
Capillary column requires smaller amounts of injected analytes compared to
packed columns.
The vaporization chamber is typically heated 50 °C above the lowest boiling point
of the sample and subsequently mixed with the carrier gas to transport the sample
into the column.
2- Sample injection system
a- Split injection
The gas flow passes through the septum
purge and the split vent.
some of the sample injected into the injector
by the sample syringe will get vaporized and
escape through the split vent.
When the split ratio is high, the rate of the
injected sample being introduced into the
column decreases as well as the sensitivity
Ratio =
b- Splitless Mode
The split line is now closed
(closed split/splitless valve).
The largest part of the sample
has been introduced into the
column, usually 10-40 secs.
Sample is introduced onto the
column during the entire
splitless time.
c- On-Column Injection
The majority of the
sample is introduced into
the capillary column.
It is recommended that
the column length be 25
m or more.
Short columns of 15 m or
less are often difficult to
use due to low set
pressure.
