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CHROMATOGRAPHY
Chromatography is a chemical technique for separating mixtures of coloured chemicals. This technique is
important in biology as well as chemistry; it is also used by forensic scientists.
Introduction
We all know that green plants are green because they contain chlorophyll; we know that chlorophyll is
green. Hang on; why are plants different shades of green if they all contain the same green pigment? Well,
perhaps it is not quite as simple as you were told by your biology teacher. In fact there are several different
kinds of chlorophyll and some other photosynthetic pigments in green plants. So exactly what colour they
are depends upon which types of chlorophyll and other pigments their leaves contain.
What has all this to do with chromatography?
What is chromatography and what can we do with it?
Well, for a start we could separate the different pigments in leaves and find out a bit more about how
photosynthesis works. Leaves contain a mixture of two or more of the following pigments: chlorophyll a,
chlorophyll b, chlorophyll c, xanthophyll, carotene, phaeophytin and other pigments. The leaves of different
plants contain different pigments, hence their different colours. We could also use chromatography to
separate the different pigment in writing ink: a forensic scientist might do this to find out if all the writing on
a cheque was written with the same pen.
We can even use chromatography to separate mixtures of colourless chemicals: it is possible to use the
technique to separate the amino-acids produced when a protein is digested. Once we have separated the
mixture it is necessary to stain the amino-acids using a coloured chemical. Ninhydrin is used; unfortunately
this is not a very nice chemical. Smoke cigarettes if you must but don't use carcinogenic chemicals without
taking the proper precautions.
Thin-layer chromatography
In analytical chemistry, technique for separating dissolved chemical substances by virtue of their differential
migration over glass plates or plastic sheets coated with a thin layer of a finely ground adsorbent, such as
Analytical Chemistry Lab
Nasir Mehmood
silica gel or alumina, that is mixed with a binder such as starch or plaster of paris. The technique, which has
become a standard analytical tool in food and pharmaceutical laboratories, is especially useful for
separating the components of naturally occurring substances, notably those found in animal and vegetable
tissues called lipids and the volatile and fragrant components of plants and flowers known as terpenes.
TLC is a liquid-solid adsorption technique where the mobile phase ascends the thin layer of stationary
phase coated onto a backing support such as glass by capillary action. There is a similar relationship to
column chromatography where the solvent travels down through the columns adsorbent. The similar
relationship allows TLC to be a rapid method for determining solvent composition for preparative
separations.
2. Affinity
To achieve a separation, the sample must have a relatively equal affinity for the solvent and the packing
material. If the sample has a higher affinity for the stationary phase than the solvent, the sample will remain
at the origin (Rf value will be too low).
3. Resolution
Resolution is improved by optimizing the affinity between sample, solvent, and support. The optimum
solvent for separating two or more compounds will maximize the difference in the compounds. Most TLC
Analytical Chemistry Lab
Nasir Mehmood
and preparative mobile phase systems contain a polar solvent and a chromatographically dissimilar lesspolar solvent. As a guide for method development, a substitution in the polar solvent often results in a
change in resolution, while a change in the less-polar solvent results primarily in a change in Rf of the
sample components. The table below shows some common tendencies of various functional groups to
adsorb onto the silica.
Affinity of Functional Groups for Silica Gel
http://www.purchon.com/chemistry/chromatography.htm
http://www.chromatography-online.org/
http://www.britannica.com/EBchecked/topic/592124/thin-layer-chromatography
http://www.discoverysciences.com/uploadedFiles/Site_for_Catalog_2008/prep-flashtlc/Thin_Layer_Chromatography/Introduction/tlc_introduction.pdf.