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Principles of chromatography
The aim of chromatography is to separate a mixture into its constituents.
Stationary phase and mobile phase are the two key components of

This is either the chromatography paper or a thin layer chromatography

plate (TLC Plate).

The chromatography paper is made of cellulose and the TLC Plate is made
of a sheet of plastic coated with silica gel or aluminium hydroxide.

In each case there are free OH groups pointing outwards for mobile


This is the solvent for the biological molecules.

At a simple level we can use water for polar molecules and ethanol for
non-polar molecules.

The mobile phase flows through and across the stationary phase carrying
the biological molecules with it.

What happens
As the solvent travels up the TLC plate or paper, the components of the solution
mixture travel with it. As the solvent reaches the top of the plate/ paper some
components are travelling slowly and some are travelling quickly. This results in
components being at different positions on the plate.

We can use the relative distance travelled to help identify the pigments


Rf value can be calculated by measuring the distance from the pencil line
to the centre of the spot of pigment (x) and the distance from the pencil
line to the solvent front (y).

Knowing Rf values of pigments allows us to identify them.

Sometimes with colourless molecules it is difficult to see where they finish. Using
thin layer chromatography there are solutions:
1. ULTRAVIOLET. Thin layer chromatography plates have a chemical which
fluoresces under uv light. Looking at the plates under uv light you will
notice it will all glow except for the places where the spots have travelled

2. NINHYDRIN. To see amino acids allow the plate to dry and then spray
Ninhydrin. This binds with the amino acids which become visible as brown/
purple spots.
3. IODINE. Allow the plate to dry and place in an enclosed container few
iodine crystals. Iodine forms a gas which then binds to the molecules in
each of the spots

How does it work

The speed at which the molecules move along the paper depends on their
solubility in the solvent and their polarity and in the case of chromatography
paper it may also depend on the size of the paper.
Exposed OH groups make the surface of the paper/ plate very polar allowing
them to form H bonds alongside other dipole interactions. A highly polar solute
will tend to stick to the surface and would move more slowly whereas a non-polar
solvent will travel very quickly up the plate.
If 2 molecules travel up at exactly the same speed, we could use different
solvents or change the PH to try and separate the molecules.
How is the chromatography used
Thin layer chromatography is commonly used to monitor the progress of
reactions as it works quickly. It is also used to test the urine samples of athletes
for illegal drugs and for contaminants in the food.

Separating amino acids using thin layer

TLC is a technique used to separate individual components of a mixture. The
technique is used to separate and identify a mixture of amino acids in solution.
The mobile phase picks up the amino acids and moves through the stationary
phase and the amino acids are separated.
In the stationary phase a thin layer of silica gel is applied to the rigid surface.
Amino acids are then added to one end of the gel. This end is submerged into
the organic solvent and the organic solvent then moves through the silica gel.
The rate at which the different amino acids in the organic solvent moves
depends on the interactions they have with the silica in the stationary phase and
their solubility in the mobile phase. This results in different amino acids moving
different distances in the same period of time and causes them to separate out
from each other.

Inorganic ions

Increases the rigidity of the teeth, bones and cartilage.

It is a component of the exoskeleton of crustaceans

It is important in clotting blood and muscle contraction

It is the activator for several enzymes such as lipase

It stimulates muscle contractions

Regulates permeability of the cell membranes and the transmission of nerve


It is also important for cell wall development and middle lamella formation


Involved in the regulation of the osmotic pressure, control of water levels in body
fluid and maintenance of the PH

Affects the absorption of the carbohydrates in the intestines and the water in the

Contributes to nervous transmission and muscle contraction

Constituent of vacuoles in plants helping it maintain turgidity


Involved in the control of water levels in body fluid and maintenance of the pH

Assists active transport of material

Involved in the synthesis of glycogen and protein and the breakdown of glucose

Contributes to nervous transmission and muscle contraction

Component of vacuoles in plants helping to maintain turgidity


Involved in Photosynthesis and respiration

Involved in transport of oxygen and carbondioxide in the blood

Involved in the regulation of the blood PH


A component of amino acids, proteins, vitamins, chlorophyll and nitrogen cycle

Some hormones are made of protein ie insulin

An essential component of nucleic acids

Involved in the maintenance of PH in our body