Nasir Mehmood

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.

Principles and Practice of Chromatography

Chromatography, although primarily a separation technique, is mostly employed in chemical analysis. Nevertheless,
to a limited extent, it is also used for preparative purposes, particularly for the isolation of relatively small amounts of
materials that have comparatively high intrinsic value. Chromatography is probably the most powerful and versatile
technique available to the modern analyst. In a single step process it can separate a mixture into its individual
components and simultaneously provide an quantitative estimate of each constituent. Samples may be gaseous,
liquid or solid in nature and can range in complexity from a simple blend of two entantiomers to a multi component
mixture containing widely differing chemical species. Furthermore, the analysis can be carried out, at one extreme,
on a very costly and complex instrument, and at the other, on a simple, inexpensive thin layer plate.

How does chromatography work?

The solvent rises up the chromatography paper (blotting paper) by capillarity. When the solvent reaches the
"spot" it dissolves the mixture of coloured chemicals. There is now a solution; this is a mixture of solutes
dissolved in the solvent. The molecules of these different chemicals are all different sizes. The simple
explanation is that the smallest solute molecules travel almost as quickly as the solvent molecules and so
get carried to the top of the chromatogram. The largest solute molecules travel very slowly and stay near
the bottom. So some of the coloured chemical travel further than others. If you are doing this with aminoacids you will not see anything happen until the end of the experiment when you stain it with ninhydrin.

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
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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.

Steps for Method Development

Choose Stationary Phase
Choose a scalable TLC plate, preferably that has an identical media as the preparative column. Choose
between normal and reverse phase based on sample polarity and solubility.
Choose a Mobile Phase
Criteria for Choosing a Preparative Solvent
Solubility
Affinity
Resolution
1. Solubility
Many solvent systems provide the minimal solubility for the sample, but to elute a sample from a column
the mobile phase must have a greater solubility for the sample, as the sample concentration is usually very
high. When possible, it is best to dissolve the sample in the mobile phase. The first step in solvent selection
is determination of the solubility of the sample. The desired mobile phase would provide the greatest
solubility, while providing affinity for the sample on the stationary phase.
Solvent Solubility Screening Table

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
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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

Perform TLC Analysis

Look up the affinity for the type of compound as well as the solvent strengths to find a starting point for
method development or look up a reference from a similar structure, and then adjust the mobile phase
composition to adjust the Rf. It is common to try 36 solvent systems for the first round of method
development. Review the results after visualization and adjust the Rf if necessary, increase the separation
and evaluate visualization techniques to make sure you are seeing all necessary compounds.
Optimizing TLC Separations for Preparative Separations
The optimum separation of compounds by TLC is usually achieved when Rf values are between 0.30.5.
Rf = Distance from origin to center of spot Distance from origin to solvent front Generally, adjusting the
compounds Rf between 0.30.5 is done first for a TLC separation. For scale-up to preparative separations,
the TLC solvent systems polarity must be decreased to lower the Rf between 0.150.35. This Rf range is
optimal for a preparative separation, in terms of sample load, resolution, residence time, and solvent usage.
REFEERENCES

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.

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