2. Chromatography is a physical method of separation in which
components to be separated are distributed between two
phases, one of which is stationary phase while the other
mobile phase move in a definite direction. The stationary
phase may be a solid or a liquid supported on a solid or a gel.
The mobile phase may be gaseous or liquid.
3. Invention of Chromatography
Mikhail Tswett
Russian Botanist
(1872-1919)
Mikhail Tswett invented
chromatography in 1901
during his research on
plant pigments.
He used the technique to
separate various plant
pigments such as
chlorophylls, xanthophylls
and carotenoids.
4. Original Chromatography Experiment
Later
Start: A glass
column is filled
with powdered
limestone
(CaCO3).
End: A series of
colored bands is
seen to form,
corresponding to
the different
pigments in the
original plant
extract. These
bands were later
determined to be
chlorophylls,
xanthophylls and
carotenoids.
An EtOH extract
of leaf pigments
is applied to the
top of the column.
EtOH is used to
flush the pigments
down the column.
5. Chromatography: (Greek = chroma “color” and
graphein “writing” ) Tswett named this new technique
chromatography based on the fact that it separated the
components of a solution by color.
Common Types of Chromatography
Tswett’s technique is based on Liquid Chromatography.
There are now several common chromatographic
methods. These include:
Paper Chromatography
Thin Layer Chromatography (TLC)
Liquid Chromatography (LC)
High Pressure Liquid Chromatography (HPLC)
Ion Chromatography
Gas Chromatography (GC)
6. • Gas chromatography (GC) is one of the popular
chromatography techniques to separate volatile compounds or
substances, based on partitioning analytes between two
immiscible phases: gaseous mobile phase (Carrier gas) and a
stationary solid or immobilized liquid phase (packed or
hollow capillary column).
7.
8.
9.
10. The principle of gas chromatography
Components in the mixture are distributed between two phases, one of which
is a stationary phase, and the other is a mobile phase gas, or carrier gas, that
carries the mixture through the stationary phase. Compounds in the mobile
phase interact with the stationary phase as they pass through. Due to the
differences in properties and structures of each component, the size and
affinity of each interaction with the stationary phase are different. Therefore,
under the same driving force, the retention time of different components
differs in the column, thus moving out of the column in different orders.
11. The sample is first vaporized by a heated inlet system to be
passed through a gaseous carrier into GC column. The passed
analytes adsorbed on the stationary phase of the GC column,
the adsorbed analytes eluted by applying heating program (the
stronger adsorption of an analyte, the higher heat required for
its elution). Therefore, GC is suitable for analysis of volatile
(or semi-volatile) and a thermally stable analyte.
14. Particular requirements
Carrier gas
Flow regulators & flow meters
Injection devices
Columns
Temperature control devices
Detector
Recorders & Integrators
15.
16. Requirements of carrier gas
Inertness
Suitable for the detector
Highly purity
Easily available
Cheap
Should not cause the risk of fire
Should give best column performance
17.
18. Advantages of GC
Speed
The entire analysis is completed less than half an hours. The use of gas
as the moving phase has the advantage of rapid equilibrium between
the moving and stationary phases and allows high carrier gas velocities
to be employed. Separations requiring only seconds have been
reported, however, analysis time of minutes duration is more common
in GC.
Resolution
Separation of some compounds such as methyl esters of stearic, oleic
and linoleic acids by other techniques is extremely difficult or
impossible. These compounds vary only in degree of unsaturation. By
using selective solvents, however, GC can provide resolution impossible
by distillation or other techniques.
19.
20. • Small sample consumption and high detection sensitivity: 1 ml of gas
sample consumption, 0.1 µl of liquid sample consumption, a few mg
of solid sample consumption. Proper detectors can detect impurities
in the tens to a few parts per million.
• Wide range of applications
About disadvantages of GC:
It can only be used to analyze volatile substances.
Editor's Notes
http://en.wikipedia.org/wiki/Mikhail_Tsvet
The method was described on 30 December 1901 at the XI Congress of Naturalists and Physicians (XI съезд естествоиспытателей и врачей) in St. Petersburg. The first printed description was in 1903, in the Proceedings of the Warsaw Society of Naturalists, biology section. He first used the term "chromatography" in print in 1906 in his two papers about chlorophyll in the German botanical journal, Berichte der Deutschen botanischen Gesellschaft. In 1907 he demonstrated his chromatogaph for the German Botanical Society.
Tsvet's work was ignored for several decades because of diverse reasons: the tragic events in Russia at the beginning of the 20th century, the fact that Tsvet originally published only in Russian (what made his results inaccessible to western scientists) and an article denying Tsvet's findings. Willstater and Stoll tried to repeat Tsvet's experiments but because they used an aggressive adsorbent (what destroys the chlorophyll's) were not able to do so. They published their results and Tsvet's chromatography method went into oblivion. It was recollected 10 years after his death thanks to German scientist Edgar Lederer and Austrian biochemist Richard Kuhn and the work of Martin and Synge.
