2.3 Paper
Chromatography or Thin Layer
Chromatography
This method of separation is used to see
what coloured materials make up e.g. a food dye analysis (e.g.
smarties), separating the different coloured dyes in an ink e.g.
felt tip pen inks. Chromatography can be used to identify substances and
check on the purity of a substance.
For example you could then investigate whether a pen ink
is made up of one or more colours by paper chromatography.
The different food colourings in confectionary products e.g. in the icing
top of a cake, the sugar coating on smarties etc. can all be separated and
identified using paper chromatography.
The coloured material mixture to be
separated e.g. a food dye (6 on the diagrams below) is dissolved in a solvent like ethanol
('alcohol') and carefully spotted onto
chromatography paper or a thin layer of a white mineral material on a
glass sheet (immobile or stationary phase).
Alongside it are spotted known colours
of pure dyes on the
reference 'start line' (1-5), which is drawn in pencil so it doesn't 'run or
smudge' - see the diagram below.
The paper is carefully
dipped into the solvent (mobile phase) and suspended so the start line (baseline) is above the
liquid solvent, otherwise all the spots would dissolve in the solvent!
The solvent is absorbed into the paper and rises up
it as it soaks into the paper.
The solvent may be water (aqueous solvent) or an organic liquid
non-aqueous solvent like an alcohol
(e.g. ethanol, butanol) or a hydrocarbon (e.g. hexane).
The technique and principles of
how to do paper chromatography is illustrated below.
A
DIAGRAM TO ILLUSTRATE the principles of PAPER CHROMATOGRAPHY
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(a) At
the start mark a baseline in pencil - this mark won't dissolve or run. Make sure it
is above the
surface of the solvent, so as not to dissolve the spots directly.
You then carefully put spots of the mixtures and standards (known
materials) onto the baseline. so we start with
an even line of solute chemical spots e.g. dyes or any coloured
compound. The prepared
chromatogram paper is carefully placed in the solvent so the
baseline and spots are above the solvent - and the solvent
(grey) begins to move up the paper. |
(b)
As the
solvent seeps into the paper and moves (spreads) up the paper
it dissolves the ink spots and the colours
begin to separate out.
It is best done in a larger covered beaker so
the solvent doesn't evaporate into the laboratory! |
(c) When the solvent is
near the top of the paper, the paper is removed and allowed to dry
before examination and measurement of the
Rf values of all the dyes. The final result is called the
chromatogram. If any
of the dyes are insoluble in the solvent, they just stay on the
start line and no analysis of them can be done.
The 'finish line' is the highest point
reached by the solvent front. |
(d)
How do we measure an Rf value from the resulting chromatogram? Its quite simple to measure a
reference value ratio (Rf) for a dye colour.
e.g.
for the green dye spot,
S might be 7.2 cm,
D might be 5.3 cm, so
Rf
= D/S = 5.3/7.2 = 0.74 |
For best results:
(i) try different solvents eg water
or ethanol ('alcohol'), propanone ('acetone') or butanol.
(ii) If you can enclose the whole
system in a large glass container with a lid on.
This reduces evaporation
of the solvent.
(iii) The fastest moving spots should
be able to move at least 5 cm to get a reasonably accurate Rf value.
(iv) However, if any of the dyes are
insoluble in the solvent, they just stay on the start line and no
analysis of them can be done because there is no separation to make an Rf
measurement (see below) to compare with known standard dye colours.
For
accurate work the distance moved by the solvent is marked on carefully with a
pencil and the distances moved by each 'centre' of the coloured spots is
also measured.
These can be compared with known substances BUT if so, the
identical paper and solvent must be used (See Rf values below and
diagrams above).
The Rf values is the ratio of how far the spot
travels (D on diagram) relative to the distance moved by the solvent front (S on
the diagram) and must have values of >0 and <1 to be of any use.
|
distance moved by
dissolved substance spot
(D) |
Rf
= |
------------------------------------------------------------------------------ |
|
distance moved by solvent front
(S) |
Each dye has its own reference values Rf
for a particular set of experimental conditions
Chromatography Rf vales depend
on the substance, the type of paper (stationary phase) AND the solvent
(mobile phase).
To get consistent chromatography results you must use
identical conditions, including temperature, and identical chemical
reagents. These criteria is an example of 'fair testing' methodology.
You
may have to experiment with solvents to ensure the best separation of the
components for the most accurate analysis.
Generally speaking if
spots match up, it shows what is present, if no matching spot, that
dye wasn't present in the mixture.
However, (i) sometimes you
get a match in one solvent and (ii) not another - this means (i) is
a coincidence, because the reference dye and component mixture dye
spots should match up whatever the component - need to think
carefully about this point.
THEORY of thin layer/paper
chromatography e.g. for a series of dyes
In chromatography the separation
of the components in a mixture depends on their distribution between the
mobile phase and the stationary phase.
Due to different
solubility's of the coloured chemicals in the solvent AND different
strength's of molecular 'adhesion' attraction to the paper, some colours move more
than others up the paper faster, so effecting the separation of the different
coloured molecules.
So the effectiveness of
the separation depends on how soluble the chemical is in the solvent
and how strongly the chemical is attracted to the paper.
Different solvents would be
tested to see which was the most effective in separating the
materials.
The distance the substance
travels up the paper depends on:
(i) How soluble the
substance is in the solvent - the mobile phase.
(ii) How strongly
attracted the substance is to the paper - the stationary
(immobile) phase - this is an intermolecular attraction.
Since these both differ from
substance to substance, a separation takes place, the most
soluble in the solvent or the least strongly attracted to the
paper, will travel the farthest up the paper to give the largest
Rf value.
Each solute dye is distributed
between the paper and the solvent (the two phases) and there is
constant movement of molecules between the stationary and mobile phases.
At any instant there is a
dynamic equilibrium of dye concentration between the phases, meaning the number of dye molecules
moving from the paper to the solvent equals those moving from the
solvent to the paper.
Each dye has a different
solubility and different strength of attraction to the paper - its
all about interaction with the mobile solvent phase (ease of
dissolving) and intermolecular attractive forces between the paper
and the dye molecules.
However, if one dye is more
strongly held by the paper (stationary phase), and the less soluble it
is in the mobile phase solvent, its progress up the paper is slowed down, hence the separation of the colours.
The final result is the vertical
separation of the spots up the paper which is now referred to as the chromatogram.
Any colour which
horizontally
matches another is likely to be the same molecule i.e. red (1 and
6),
brown (3 and 6) and blue (4 and 6) match, showing these three coloured
substances are all in the food
dye (6), the dye that's being analysed by paper chromatography.
In the diagram, think of dye
(colouring) spots 1 to 5 as the known food dye colours, therefore food colouring dye 6
must be a mixture of three dyes, that is food colourings 1, 3 and 4 because these are the spots that
line up horizontally with known standard samples. Dye 6 is composed of a
mixture of red (1), brown (3) and blue (4) dyes.
Note that 1. to 5. give one
spot each AND that's what you would expect for a pure
compound. In fact it would be useless to have impure standards!
Suppose the solvent reached
8.0 cm from the baseline and for the coloured substances e.g
red spot 1.5 cm, Rf =
1.5/8.0 = 0.19; green spot 4.5 cm, Rf =
4.5/8.0 = 0.56; blue spot 7.5 cm, Rf =
7.5/8.0 = 0.94
The distance a substance
moves, compared to the distance the solvent front moves (top of grey area on
2nd diagram) is called the reference or Rf value and has a
value of 0.0 (not moved, not soluble, no good), to 1.0 (too soluble - no good either), but
Rf ratio values between 0.1 and 0.9 can be useful for analysis and
identification of coloured dye-like materials.
Rf =
distance moved by dissolved substance (solute) / distance moved by solvent
front
Rf values are used in
many analytical situations to identify substances e.g. forensic
science - analysing evidence samples, dye analysis in the food industry,
food additives, testing for drugs, biochemistry e.g. analysing amino
acids in a protein structure
Some technical terms: The
substances (solutes) to be analysed must dissolve in the solvent,
which is called the mobile phase because it moves. The paper or thin
layer of material on which the separation takes place is called the stationary or immobile phase because it doesn't move.
It is possible to analyse
colourless mixture of chemicals if the 'spots' can be made coloured
by some further chemical or light treatment e.g.
(i) protein
can be broken down into amino acids, separated with paper
chromatography and coloured purple by a chemical
reagent
called Ninhydrin.
Each protein has its unique
Rf value for a particular solvent.
(ii) Many
colourless
organic molecules fluoresce when ultra-violet light is shone on them, so the spots show up under uv
light.
These extra reagents are called
locating
agents and enable Rf values to be measured and amino
acids and lots of molecules to be identified.
If a substance is pure, only one spot
will appear on the chromatogram, impurities may show up as other faint
spots.
Thin
layer chromatography (t.l.c, in chemistry, is not 'tender loving care'!) is where a layer of paste is thinly and evenly
spread on e.g. a glass plate. The paste consists of the solid immobile phase
like aluminium oxide dispersed in a liquid such as water (thick paste and
dried out) or silica gel. The mobile phase or solvent is just the same as
paper chromatography e.g. ethanol, other alcohol etc.
Important note: If the starting spot moves up
the paper and remains as a single spot, it means that substance must
be pure and not a mixture. If it was a mixture, then at least two
spots would be seen.
Gas-liquid
chromatography is described further down the page
For more on the chromatography of amino
acids see: GCSE
notes
AMINO ACIDS and natural
polymers
Advanced A Level Notes
Amino acids - molecular
structure, preparation and reactions
A plant material extraction
process using chromatography The experiment described below simulates one way
in which drug companies extract plant material to develop products in the
pharmaceutical industry. 1. Take some suitable plant material and crush it with a pestle and mortar.
2. Scrape the crushed plant material and gently
heat with a small volume of suitable solvent like water or alcohol to
dissolve and extract some of the soluble coloured plant material.
3. Filter off the residue to give a clear but
coloured concentrated solution.
4. Take the solution and spread it along the start
line of some chromatography paper (diagram A).
5. Dip and suspend the chromatography paper into
a suitable solvent in a covered container so the start line is above the
solvent surface and let the solvent be absorbed by the chromatography paper
and move upwards (diagram B).
6. When the solvent front has reached near the
top of the paper, stop (diagram C) and hang up the chromatogram to dry.
7. You can then separate the mixture
'physically' by cutting strips off for each band of coloured material that
has separated out on the chromatogram (diagram D).
8. You can then extract each separated product
by re-dissolving it by placing the strips in the solvent.
9. The different solutions can then be carefully
evaporated to produce the separated coloured solid materials.
Note: In practice in the pharmaceutical industry
large scale thin layer chromatography would be used.
Another
example of how to interpret a chromatogram where paper chromatography
has been used to analyse coloured materials.
Lets assume we have pure red, blue and yellow dye
molecules.
These three rise to different heights on the
chromatograms.
Three different Rf reference values (yellow > red >
blue).
By matching the spots horizontally, you can deduce:
(i) The purple ink contains red and blue dye molecules.
(ii) The green ink contains blue and yellow molecules.
(iii) The brown ink contains red, blue and yellow
molecules. |