C.A.P.E.

CHEMISTRY
What is Chromatography?
Recall the first time you were introduced to colour in art
class and you learnt of the three primary colours and the
resulting secondary colours.
Chromatography is concerned with being able to separate
mixtures into their individual components.

This can be useful for detection purposes, but may also be

used to quantify the different components.
Types of chromatography

There are many types, however, you need to be familiar

with the following
1. Paper
2. Thin layer (tlc)
3. Column
4. Gas-liquid (glc)
Principles of Chromatography

All types of chromatography have the following principles

in common.
1. There are two phases: the stationary phase and the
mobile phase
2. The separation of the mixture is due to differing
interaction of the components with the stationary phase
3. The two mechanisms which can occur are partitioning
and adsorption
What is the stationary phase?
This is the immovable phase and may either be a solid
support:
• Cellulose in the paper
• Silica or alumina

or it may be a non-volatile, viscous liquid coated unto a

solid surface
• A long-chain alkane of high boiling point on a SiO2
support
What is the mobile phase?

This is the solvent which moves through the column or

over the surface of the stationary phase and can be a
gas or a liquid.

There are different types of solvents, ranging from polar

(such as water) to non-polar (such as alkanes)

The mobile phase can be a mixture of solvents, in which

case the ratio is quoted
e.g. methanol:water (2:1)
 PARTITIONING AND ADSORPTION
Adsorption chromatography occurs when the components
of the mixture (solute molecules) are bound to the surface
of the stationary phase.

The stationary phase is a polar solid (such as silica or

alumina) and the polar solute molecules are bound to this
surface. The more polar the solute, the more strongly
bound it is to the stationary phase

As the mobile phase passes over this, the solute

molecules are eluted, from least polar to most polar in
turn.
Partitioning occurs between liquids.

If colourless chloroform is added to aqueous iodine (a

brown solution,) two layers develop, with the chloroform
layer below the aqueous layer and having a purple colour.

The iodine moves between the two layers until it reaches

an equilibrium, at which point it is in a definite ratio. This is
partitioning.

If both the stationary and the mobile phases are fluids, then
the solute will be partitioned between the two.
Solute in the mobile phase will move along with it and
elution will take place starting with those compounds
more soluble in the mobile phase and ending with those
least soluble.
 PAPER CHROMATOGRAPHY
Filter paper is used to aid in separation

Cellulose fibres contain water which acts as the stationary

phase.

A small dot of mixture is placed on the paper, which is

placed in a jar containing a shallow layer of solvent and is
sealed
 The Process of Paper
Chromatography

https://defra.jot.com/System/TmpImageUpload/Chromatogr
aphy.jpg
Methods of Analysis and Detection, Cambridge University Press
As the solvent moves up the paper, the different
components are partitioned between the stationary and
mobile phase.

Least polar molecules move ahead of the more polar

molecules.

Can be used to separate components of black pen ink

http://www.columbia.edu/cu/biology/courses/c2005/handouts/chrom2.4.gif
Separation of black ink using Paper Chromatography

http://wps.prenhall.com/wps/media/objects/165/169061/GIFS/AAAVBCN0.JPG
Retention factor (also called
retardation factor) : Rf
• The movement of a solute
relative to the movement of
the solvent front
 THIN LAYER CHROMATOGRAPHY

Stationary phase is a thin layer of silica (SiO2) or alumina

(Al2O3) coated unto a plastic or glass support.

Setup is similar to that of paper chromatography, but this

utilises adsorption and not partition.

Useful for separating colourless components which can be

detected by use of appropriate chemicals or techniques
(visualising agents)
 Thin layer chromatography used to
separate the components of chlorophyll

http://en.wikipedia.org/wiki/Chromatography
Visualising agents
1. Iodine crystals may be
added to the solvent and
as iodine vapour
evolves, this is
accumulated on the
spots of the separated
solute.
Result is dark brown
spots on a yellow
background

http://www.chem.ucsb.edu/~kalju/chem110L/public/TLC_2004.png
1. Spraying amino acids with ninhydrin causes them to
appear lilac in colour

http://www.chemguide.co.uk/analysis/chromatography/thinlayer.html
1. Visualising colourless solute using UV light
Advantages of TLC over Paper Chromatography

1. TLC is faster (approximately three times as fast)

2. TLC works well with very small samples
3. The thin layer can be made from different solids, so a
wide range of mixtures can be separated by carefully
selecting the mobile and stationary phases
4. TLC can be used to quickly select the best conditions for
larger scale separation (such as by column
chromatography)
Application of TLC

Wide range of applications

1. Pesticide analysis: separation of chlorinated insecticides
2. Chemical Forensics: separation and detection of
alkaloids (e.g. morphine and opium) and cannibis also
used as a screen for explosives
3. Drug testing: Detection of steroids
4. Pharmaceutical: Determination of the purity of new drugs
(quality control purposes)
 COLUMN CHROMATOGRAPHY

Stationary phase is silica (SiO2) or alumina (Al2O3) placed

in a column. (note, this is a polar phase)

The mixture is placed above the stationary phase and

eluted with the mobile phase under gravity or under
pressure (flash chromatography.)

Can be used to separate quantities ranging from

micrograms to kilograms and is based on adsorption
mechanism
The aim is to achieve good separation by carefully
choosing solvent systems which allow one component to
move through the column faster than the other
components.

Stationary phase is polar and will bind strongest to the most

polar component of the mixture

Mobile phase ranges from non-polar through to polar, with

a different solvent mixture being used to elute successively
polar components
Useful for purification purposes as mixtures can be separated into individual
components and be collected in fractions.

http://www.chemguide.co.uk/analysis/chromatography/column.html
Application of Column Chromatography

1. Purification of natural products

2. Purification of pharmaceuticals
3. Purification of chemical reactions
4. Separation of dyes and pigments
 GAS-LIQUID CHROMATOGRAPHY

Used to separate and identify small quantities of gases, liquids

and volatile solids

Vapourised sample is carried by a mobile phase (inert gas)

over a stationary phase (non-volatile liquid on a solid support)

Partitioning of the components of the sample occurs and they

move through the column based on
• their volatility
• their relative solubility in the mobile and stationary phases
Diagram of a typical Gas-Liquid Chromatograph
The components leave the column after a definite time
period.
Retention time: time between the injection of the mixture
and the centre of the peak corresponding to a component

Area under a component peak is proportional to the amount

of that component in the mixture

Useful for detection, determination and quantification of

compounds and is used in conjunction with mass
spectrometry for confirmation of the components eluted.
(GC-MS)
Application of GLC

1. Pesticide analysis
2. Analysis of crude oil
3. Environmental analysis: detection of pollutants
4. Determination of the components of natural oils and
flavours
1. A particular component with a low affinity for the stationary phase will
generally move a ________ distance along the stationary phase than a
component with a high affinity for the stationary phase in a
chromatography experiment.
A. longer
B. shorter

2. Let Ds be the distance the solvent travels, and let D1 be the distance
component 1 travels on the chromatogram. The retention factor for
component 1 is defined to be:
A. D1 x Ds
B. D1/Ds
C. 1/( D1 x Ds)

3. A beaker will be used as a "developing jar" in an experiment. When the

TLC plate is set in this beaker, the solvent in the beaker must be:
A. above the pencil line used to guide the spotting of samples
B. deep enough to cover the entire TLC plate
C. deep enough to come about halfway up the TLC plate
D. below the pencil line used to guide the spotting of samples