Chromatography

Is a technique used to separate and

identify the components of a mixture.
Works by allowing the molecules present in
the mixture to distribute themselves between
a stationary and a mobile medium.

Molecules that spend most of their time

in the mobile phase are carried along
faster.

PAPER CHROMATOGRAPHY
Chromatography is used to separate
mixtures of substances into their
components.
All forms of chromatography work on
the same principle.

 They all have

A stationary phase (a solid, or a liquid supported
on a solid) and
A mobile phase
(a liquid or a gas).
flows through the stationary phase and
carries the components of the mixture with it.
Different components travel at different rates.
In paper chromatography, the stationary phase is a
very uniform absorbent paper. The mobile phase is
a suitable liquid solvent or mixture of solvents

Producing a paper chromatogram

paper chromatography
separates out mixtures of
coloured dyes - for example,
the dyes which make up a
particular ink.

 Suppose you have three blue pens and you

want to find out which one was used to
write a message.
Samples of each ink are spotted on to a
pencil line drawn on a sheet of
chromatography paper.
Some of the ink from the message is
dissolved in the minimum possible amount
of a suitable solvent, and that is also
spotted onto the same line.
In the diagram, the pens are labelled 1, 2
and 3, and the message ink as M.

 The paper is suspended in a container with a shallow

layer of a suitable solvent or mixture of solvents in it.
It is important that the solvent level is below the line
with the spots on it.
Sometimes the paper is just coiled into a loose
cylinder and fastened with paper clips top and
bottom. The cylinder then just stands in the bottom
of the container.
The container is then covered to make sure that the
atmosphere in the beaker is saturated with solvent
vapour. This stops the solvent from evaporating as it
rises up the paper.

 As the solvent slowly

travels up the paper,
the different
components of the ink
mixtures travel at
different rates and the
mixtures are
separated into
different coloured
spots.

from the final

chromatogram

the pen that

wrote the
message
contained the
same dyes as pen
2.
pen 1 contains a
mixture of two
different blue
dyes - one of

Rf values
Some compounds in a mixture travel
almost as far as the solvent does;
some stay much closer to the base line.
The distance travelled relative to the
solvent is a constant for a particular
compound as long as everything is keept
constant (the type of paper and the exact
composition of the solvent)
The distance travelled relative to the
solvent is called the Rf value.
it can be worked out using the formula:

 You are making the assumption that if

you have two spots in the final
chromatogram which are the same
colour and have travelled the same
distance up the paper, they are most
likely the same compound.
It isn't necessarily true of course - you
could have two similarly coloured
compounds with very similar Rf values.

What if the substances you are

interested in are colourless?
In some cases, it may be possible to make the
spots visible by reacting them with something
which produces a coloured product.
A good example of this is in chromatograms
produced from amino acid mixtures.
Suppose you had a mixture of amino acids and
wanted to find out which particular amino acids
the mixture contained.
For simplicity we'll assume that you know the
mixture can only possibly contain five of the
common amino acids.

 A small drop of a solution of the mixture is placed on

the base line of the paper, and similar small spots of
the known amino acids are placed alongside it.
The paper is then stood in a suitable solvent and left
to develop as before.
In the diagram, the mixture is M, and the known
amino acids are labelled 1 to 5.
The position of the solvent front is marked in pencil
and the chromatogram is allowed to dry and is then
sprayed with a solution of ninhydrin.
Ninhydrin reacts with amino acids to give coloured
compounds, mainly brown or purple.

The left-hand diagram shows the paper after the

solvent front has almost reached the top. The spots are
still invisible. The second diagram shows what it might
look like after spraying with ninhydrin.

 There is no need to measure the Rf

values (direct comparison)
In this example, the mixture contains
the amino acids labelled as 1, 4 and
5.

if the mixture contained amino

acids other than the ones we have
used for comparison:
There would be spots in the
mixture which didn't match
those from the known amino
acids.
Re-run the experiment using
other amino acids for
comparison.

Two way paper chromatography

Gets around the problem of separating out
substances which have very similar Rf values.
You can perfectly well do this with colourless
compounds - but you have to use quite a lot of
imagination in the explanation of what is going
on!
This time a chromatogram is made starting from
a single spot of mixture placed towards one end
of the base line.
It is stood in a solvent as before and left until the
solvent front gets close to the top of the paper.

In the diagram,
the position of
the solvent front
is marked in
pencil before the
paper dries out.
This is labelled as
SF1 - the solvent
front for the first
solvent. We shall
be using two
different solvents.

 The large central spot in the chromatogram is partly blue

and partly green.
Two dyes in the mixture have almost the same R f values.
They could equally well, of course, both have been the
same colour - in which case you couldn't tell whether
there was one or more dye present in that spot.
What you do now is to wait for the paper to dry out
completely, and
then rotate it through 90 , and develop the
chromatogram again in a different solvent.
It is very unlikely that the two confusing spots will have
the same Rf values in the second solvent as well as the
first, and so the spots will move by a different amount.

 The next diagram

shows what might
happen to the
various spots on the
original
chromatogram.
The position of the
second solvent front
is also marked.

 You wouldn't, of
course, see
these spots in
both their
original and final
positions - they
have moved!
The final
chromatogram

 Two way chromatography has completely

separated out the mixture into four distinct spots.
If you want to identify the spots in the mixture, you
obviously can't do it with comparison substances
on the same chromatogram as we looked at earlier
with the pens or amino acids examples. You would
end up with a meaningless mess of spots.
You can, though, work out the Rf values for each of
the spots in both solvents, and then compare these
with values that you have measured for known
compounds under exactly the same conditions.

The essential structure of paper

Paper is made of cellulose fibres, and
cellulose is a polymer of glucose.
The polymer chains have -OH groups
sticking out all around them.
To that extent, it presents the same
sort of surface as silica gel or
alumina in thin layer
chromatography.

PC using a non-polar solvent

Suppose you use a non-polar solvent such as hexane to develop your
chromatogram.
Non-polar molecules in the mixture that you are trying to separate will have
little attraction for the water molecules attached to the cellulose, and so will
spend most of their time dissolved in the moving solvent.
Molecules like this will therefore travel a long way up the paper carried by
the solvent. They will have relatively high Rf values.
On the other hand, polar molecules will have a high attraction for the water
molecules and much less for the non-polar solvent. They will therefore tend
to dissolve in the thin layer of water around the cellulose fibres much more
than in the moving solvent.
Because they spend more time dissolved in the stationary phase and less
time in the mobile phase, they aren't going to travel very fast up the paper.
The tendency for a compound to divide its time between two immiscible
solvents (solvents such as hexane and water which won't mix) is known as
partition. Paper chromatography using a non-polar solvent is therefore a
type of partition chromatography.

PC using a water and other polar solvents

A moment's thought will tell you that partition can't be the explanation
if you are using water as the solvent for your mixture. If you have water
as the mobile phase and the water bound on to the cellulose as the
stationary phase, there can't be any meaningful difference between the
amount of time a substance spends in solution in either of them. All
substances should be equally soluble (or equally insoluble) in both.
And yet the first chromatograms that you made were probably of inks
using water as your solvent.
If water works as the mobile phase as well being the stationary phase,
there has to be some quite different mechanism at work - and that must
be equally true for other polar solvents like the alcohols, for example.
Partition only happens between solvents which don't mix with each
other. Polar solvents like the small alcohols do mix with water.
In researching this topic, I haven't found any easy explanation for what
happens in these cases. Most sources ignore the problem altogether
and just quote the partition explanation without making any allowance
for the type of solvent you are using. Other sources quote mechanisms
which have so many strands to them that they are far too complicated

THIN LAYER
CHROMATOGRAPHY

Carrying out TLC

TLC is done exactly as it says - using a thin,
uniform layer of silica gel or alumina coated
onto a piece of glass, metal or rigid plastic.
The silica gel (or the alumina) is the
stationary phase.
The stationary phase for TLC also often
contains a substance which fluoresces in UV
radiation
The mobile phase is a suitable liquid solvent
or mixture of solvents

Producing the chromatogram

A particular
dye is in fact
a mixture of
simpler dyes.

 A pencil line is drawn near the bottom of the plate and a small drop of
a solution of the dye mixture is placed on it. Any labelling on the plate
to show the original position of the drop must also be in pencil. If any
of this was done in ink, dyes from the ink would also move as the
chromatogram developed.
When the spot of mixture is dry, the plate is stood in a shallow layer of
solvent in a covered beaker. It is important that the solvent level is
below the line with the spot on it.
The reason for covering the beaker is to make sure that the
atmosphere in the beaker is saturated with solvent vapour. To help
this, the beaker is often lined with some filter paper soaked in solvent.
Saturating the atmosphere in the beaker with vapour stops the
solvent from evaporating as it rises up the plate.
As the solvent slowly travels up the plate, the different components of
the dye mixture travel at different rates and the mixture is separated
into different coloured spots.

 The diagram shows the

plate after the solvent has
moved about half way up
it.
The solvent is allowed to
rise until it almost reaches
the top of the plate.
That will give the
maximum separation of
the dye components for
this particular
combination of solvent
and stationary phase.

Measuring Rf values
If all you wanted to know is how many
different dyes made up the mixture,
you could just stop there. However,
measurements are often taken from
the plate in order to help identify the
compounds present. These
measurements are the distance
travelled by the solvent, and the
distance travelled by individual spots.
When the solvent front gets close to
the top of the plate, the plate is
removed from the beaker and the
position of the solvent is marked with
another line before it has a chance to
evaporate.
These measurements are then taken:
The Rf value for each dye is then
worked out using the formula:

What if the substances you are

interested in are colourless?
There are two simple ways of getting around this problem.
Using fluorescence
the stationary phase on a TL plate often has a substance
added to it which will fluoresce when exposed to UV
radiation .
That means that if you shine UV radiation on it, it will glow.
That glow is masked at the position where the spots are on
the final chromatogram - even if those spots are invisible to
the eye.
That means that if you shine UV radiation on the plate, it
will all glow apart from where the spots are.
The spots show up as darker patches.

 While the UV is still

shining on the plate, you
obviously have to mark
the positions of the spots
by drawing a pencil
circle around them.
As soon as you switch off
the UV source, the spots
will disappear again.

Showing the spots up chemically

The chromatogram
is allowed to dry
and is then sprayed
with a solution of
ninhydrin.
Ninhydrin reacts
with amino acids to
give coloured
compounds, mainly
brown or purple.

 In another method, the chromatogram is again

allowed to dry and then placed in an enclosed
container (such as another beaker covered with
a watch glass) along with a few iodine
crystals.
The iodine vapour in the container may either
react with the spots on the chromatogram, or
simply stick more to the spots than to the rest
of the plate.
Either way, the substances you are interested
in may show up as brownish spots.

Using TLC to identify compounds

Suppose you had a mixture of amino acids and wanted to

find out which particular amino acids the mixture
contained.
For simplicity we'll assume that you know the mixture can
only possibly contain five of the common amino acids.
A small drop of the mixture is placed on the base line of the
thin layer plate, and similar small spots of the known amino
acids are placed alongside it.
The plate is then stood in a suitable solvent and left to
develop as before.
In the diagram, the mixture is M, and the known amino
acids are labelled 1 to 5.
The left-hand diagram shows the plate after the solvent
front has almost reached the top.
The spots are still invisible.
The second diagram shows what it might look like after
spraying with ninhydrin.

 There is no need to measure the Rf values because

you can easily compare the spots in the mixture with
those of the known amino acids - both from their
positions and their colours.
In this example, the mixture contains the amino
acids labelled as 1, 4 and 5.
And what if the mixture contained amino acids other
than the ones we have used for comparison? There
would be spots in the mixture which didn't match
those from the known amino acids.
You would have to re-run the experiment using other
amino acids for comparison.

How does thin layer chromatography

work?
The stationary phase - silica gel
Silica gel is a form of silicon dioxide
(silica).
The silicon atoms are joined via
oxygen atoms in a giant covalent
structure.
At the surface of the silica gel, the
silicon atoms are attached to -OH
groups.

 So, at the surface of the silica gel you have Si-OH bonds instead of Si-O-Si bonds.

The surface of the silica gel is very polar and,

because of the -OH groups, can form hydrogen
bonds with suitable compounds around it as well
as van der Waals dispersion forces and dipoledipole attractions.

 The other commonly used stationary

phase is alumina - aluminium oxide.
The aluminium atoms on the surface
of this also have -OH groups
attached.
Anything we say about silica gel
therefore applies equally to alumina.

 What separates the compounds as a

chromatogram develops?
As the solvent begins to soak up the
plate, it first dissolves the
compounds in the spot that you have
put on the base line.
The compounds present will then
tend to get carried up the
chromatography plate as the solvent
continues to move upwards.

 How fast the compounds get carried up the

plate depends on two things:
How soluble the compound is in the solvent.
This will depend on how much attraction there
is between the molecules of the compound and
those of the solvent.
How much the compound sticks to the
stationary phase - the silica get, for example.
This will depend on how much attraction there
is between the molecules of the compound and
the silica gel.

 Suppose the original spot contained two compounds - one of which can form
hydrogen bonds, and one of which can only take part in weaker van der Waals
interactions.
The one which can hydrogen bond will stick to the surface of the silica gel
more firmly than the other one. We say that one is adsorbed more strongly
than the other. Adsorption is the name given to one substance forming some
sort of bonds to the surface of another one.
Adsorption isn't permanent - there is a constant movement of a molecule
between being adsorbed onto the silica gel surface and going back into
solution in the solvent.
Obviously the compound can only travel up the plate during the time that it is
dissolved in the solvent. While it is adsorbed on the silica gel, it is temporarily
stopped - the solvent is moving on without it. That means that the more
strongly a compound is adsorbed, the less distance it can travel up the plate.
In the example we started with, the compound which can hydrogen bond will
adsorb more strongly than the one dependent on van der Waals interactions,
and so won't travel so far up the plate.

 What if both components of the mixture can hydrogen bond?

It is very unlikely that both will hydrogen bond to exactly the same
extent, and be soluble in the solvent to exactly the same extent. It
isn't just the attraction of the compound for the silica gel which
matters. Attractions between the compound and the solvent are also
important - they will affect how easily the compound is pulled back
into solution away from the surface of the silica.
However, it may be that the compounds don't separate out very well
when you make the chromatogram. In that case, changing the solvent
may well help - including perhaps changing the pH of the solvent.
This is to some extent just a matter of trial and error - if one solvent or
solvent mixture doesn't work very well, you try another one. (Or, more
likely, given the level you are probably working at, someone else has
already done all the hard work for you, and you just use the solvent
mixture you are given and everything will work perfectly!)

COLUMN CHROMATOGRAPHY
Column chromatography is often
used to purify compounds made in
the lab.

Carrying out column

chromatography
The column
In TLC, the stationary phase is a thin layer of silica
gel or alumina on a glass, metal or plastic plate.
C Cworks on a much larger scale by packing the
same materials into a vertical glass column.
Various sizes of chromatography columns are used,
and if you follow a link at the bottom of the page to
the Organic Chemistry section of the Colorado
University site, you will find photographs of various
columns. In a school lab, it is often convenient to
use an ordinary burette as a chromatography
column.

Using the column

Suppose you wanted to separate a
mixture of two coloured compounds - one
yellow, one blue.
The mixture looks green.
You would make a concentrated solution
of the mixture preferably in the solvent
used in the column.
First you open the tap to allow the
solvent already in the column to drain so
that it is level with the top of the packing
material, and then add the solution
carefully to the top of the column.
Then you open the tap again so that the
coloured mixture is all absorbed into the
top of the packing material, so that it
might look like this:

 Next you add fresh solvent to the top of the

column, trying to disturb the packing material
as little as possible.
Then you open the tap so that the solvent can
flow down through the column, collecting it in a
beaker or flask at the bottom.
As the solvent runs through, you keep adding
fresh solvent to the top so that the column
never dries out.
The next set of diagrams shows what might
happen over time.

Explaining what is happening

This assumes that you have read the explanation for what
happens during thin layer chromatography. If you haven't, follow
the very first link at the top of the page and come back to this
point afterwards.
The blue compound is obviously more polar than the yellow one it perhaps even has the ability to hydrogen bond.
You can tell this because the blue compound doesn't travel
through the column very quickly.
That means that it must adsorb more strongly to the silica gel or
alumina than the yellow one.
The less polar yellow one spends more of its time in the solvent
and therefore washes through the column much faster.
The process of washing a compound through a column using a
solvent is known as elution. The solvent is sometimes known as
the eluent.

What if you want to collect the blue

compound as well?

It is going to take ages to wash the blue compound through at the

rate it is travelling at the moment! However, there is no reason why
you can't change the solvent during elution.
Suppose you replace the solvent you have been using by a more
polar solvent once the yellow has all been collected. That will have
two effects, both of which will speed the blue compound through the
column.
The polar solvent will compete for space on the silica gel or alumina
with the blue compound.
Any space temporarily occupied by solvent molecules on the surface
of the stationary phase isn't available for blue molecules to stick to
and this will tend to keep them moving along in the solvent.
There will be a greater attraction between the polar solvent
molecules and the polar blue molecules. This will tend to attract any
blue molecules sticking to the stationary phase back into solution.
The net effect is that with a more polar solvent, the blue compound
spends more time in solution, and so moves faster.
So why not use this alternative solvent in the first place? The answer
is that if both of the compounds in the mixture travel quickly through
the column right from the beginning, you probably won't get such a

What if everything in your mixture

is colourless?
If you were going to use C C to purify
the product of an organic
preparation, it is quite likely that the
product that you want will be
colourless even if one or more of the
impurities is coloured. Let's assume
the worst case that everything is
colourless.

How do you know when the substance you

want has reached the bottom of the column?
There is no quick and easy way of doing this! What you
do is collect what comes out of the bottom of the column
in a whole series of labelled tubes.
How big each sample is will obviously depend on how big
the column is - you might collect 1 cm 3 samples or 5 cm3
samples or whatever is appropriate.
You can then take a drop from each solution and make a
thin layer chromatogram from it. You would place the
drop on the base line alongside a drop from a pure
sample of the compound that you are making. By doing
this repeatedly, you can identify which of your samples
collected at the bottom of the column contain the desired
product, and only the desired product.

 Once you know this, you can

combine all of the samples which
contain your pure product, and then
remove the solvent. (How you would
separate the solvent from the
product isn't directly relevant to this
topic and would vary depending on
their exact nature - so I'm not even
going to attempt a generalisation.)

GAS-LIQUID CHROMATOGRAPHY
GLC (often just called gas
chromatography) is a powerful tool in
analysis.

Carrying out GLC

Introduction
In GLC, the mobile phase is a gas such as
helium and the stationary phase is a high
boiling point liquid absorbed onto a solid.
How fast a particular compound travels
through the machine will depend on how
much of its time is spent moving with the
gas as opposed to being attached to the
liquid in some way.

A flow scheme for gas-liquid

chromatography

Injection of the sample

Very small quantities of the sample are
injected .
The syringe needle passes through a thick
rubber disc (known as a septum) which reseals
itself again when the syringe is pulled out.
The injector is contained in an oven whose
temperature can be controlled.
It is hot enough so that all the sample boils
and is carried into the column as a gas by the
helium (or other carrier gas).

How the column works

The packing material
There are two main types of column in GLC.
One of these is a long thin tube packed with the stationary phase;
the other is even thinner and has the stationary phase bonded to its
inner surface.
To keep things simple, we are just going to look at the packed column.
The column is typically
a) made of stainless steel
b) between 1 and 4 metres long
c) with an internal diameter of up to 4 mm.
) I t is coiled up so that it will fit into a thermostatically controlled oven.
) The column is packed with finely ground diatomaceous earth, which is
a very porous rock. This is coated with a high boiling liquid - typically a
waxy polymer.

The column temperature

The column temperature: 50C to 250C.
It is cooler than the injector oven, so that
some components of the mixture may
condense at the beginning of the column.
In some cases, the column starts off at a
low temperature and then is made
steadily hotter under computer control as
the analysis proceeds.

How separation works on the

column
One of three things might happen to a particular
molecule in the mixture injected into the column:
1) It may condense on the stationary phase.(BP >
the column temperature) =
2) It may dissolve in the liquid on the surface of the
stationary phase.(The more soluble ones will
spend more of their time absorbed into the
stationary phase)
3) It may remain in the gas phase. (the less soluble
ones will spend more of their time in the gas)
) None of these things is necessarily permanent.

partition
The process where a substance divides
itself between two immiscible solvents
because it is more soluble in one than the
other is known as partition.
a gas such as helium can't really be
described as a "solvent". But the term
partition is still used in GLC
A substance partitions itself between the
liquid stationary phase and the gas.
Any molecule in the substance spends
some of its time dissolved in the liquid and

GAS LIQUID CHROMATOGRAPHY (GLC)

Retention time The time taken for a compound to travel through the
column to the detector.
It is measured from the time the sample is injected to
the time its peak shows maximum height.
For a particular compound, the retention time depends on...
boiling point

high boiling point = long retention time

solubility in the liquid phase

greater solubility = long retention time

ANIMATION

 One of three things might happen to a particular

molecule in the mixture injected into the column:
1) It may condense on the stationary phase.(BP > the
column temperature) high boiling point means a long
retention time.
2) It may dissolve in the liquid on the surface of the
stationary phase.(The more soluble ones will spend
more of their time absorbed into the stationary phase)
High solubility in the liquid phase means a high
retention time.
3) It may remain in the gas phase. (the less soluble ones
will spend more of their time in the gas)
) None of these things is necessarily permanent.

 The lower the temperature of the column, the better the

separation, but it could take a very long time to get the compounds
through which are condensing at the beginning of the column!
On the other hand, using a high temperature, everything will pass
through the column much more quickly - but less well separated
out. If everything passed through in a very short time, there isn't
going to be much space between their peaks on the chromatogram.
The answer is to start with the column relatively cool, and then
gradually and very regularly increase the temperature.
At the beginning, compounds which spend most of their time in the
gas phase will pass quickly through the column and be detected.
Increasing the temperature a bit will encourage the slightly
"stickier" compounds through. Increasing the temperature still more
will force the very "sticky" molecules off the stationary phase and
through the column.

 The detector
There are several different types of
detector in use. The flame ionisation
detector described below is
commonly used and is easier to
describe and explain than the
alternatives.

A flame ionisation detector

In terms of reaction
mechanisms, the burning of
an organic compound is very
complicated. During the
process, small amounts of
ions and electrons are
produced in the flame. The
presence of these can be
detected.
The whole detector is
enclosed in its own oven
which is hotter than the
column temperature. That
stops anything condensing in
the detector.

 If there is nothing organic coming through from the

column, you just have a flame of hydrogen burning
in air. Now suppose that one of the compounds in
the mixture you are analysing starts to come
through.
As it burns, it will produce small amounts of ions
and electrons in the flame. The positive ions will be
attracted to the cylindrical cathode. Negative ions
and electrons will be attracted towards the jet itself
which is the anode.
This is much the same as what happens during
normal electrolysis.

In the animation below the red molecules are more

soluble in the liquid (or less volatile) than are the green
molecules.