INTRODUCTION
Artificial food colours are added to many foods
to make them look tastier and more appealing..
Most of us are familiar with their use in
sweets, and soft drinks, but fewer people
are aware of their widespread use in foods
such as sauces, cheese, butter, and various
prepared foods.
This experiment involves the use of
Solid-phase extraction (SPE).
This is a separation process by which
compounds that are dissolved or suspended in a liquid mixture are separated
from other compounds in the mixture according to their physical and chemical
properties.
Analytical laboratories use solid phase extraction to concentrate and purify
samples for analysis, which is what you will be doing.
This particular form of SPE uses reverse phase chromatography.
Reversed phase separations involve a polar or moderately polar mobile phase, in
this case water as we are using an aqueous solution, and a nonpolar stationary
phase.
The analytes of interest, in this case the colourings, are typically mid- to
nonpolar.
Binding of organic compounds from polar solutions onto these SPE materials is
due primarily to the attractive forces between the carbon-hydrogen bonds in the
analyte and the functional groups on the silica surface. These nonpolar-nonpolar
attractive forces are commonly called van der Waals forces: or dispersion
forces.
Once the compound has attached to the column, we use a nonpolar solvent to
disrupt the forces that bind the compound to the packing and thus elute it for
analysis.
Extraction of Synthetic Colours From Food
You will need
2 x 25 cm3 measuring cylinders
2 x boiling tubes
boiling tube rack
Ethanol (IDA)
Stirring rod
Cartridge with syringe attachment*
Tetrabutylammonium busulphate 000.7M
Tetrabutylammonium busulphate 0.7M
* The cartridge looks like the barrel of a syringe with a white layer about 1cm thick at the
bottom. You will also need an adaptor. This is a small, plastic device that fits tightly into the
top of the barrel of the cartridge. On the top is a small hole where the end of a syringe fits,
also quite tightly.
The idea of having such a tight fit is that it allows you to apply pressure to the liquid going
through the column using the top syringe.
Once you have got your syringe fixed in the top of the adaptor, which is in the cartridge, DO
NOT pull the plunger out. ALWAYS take the syringe out of the adaptor first. If you don’t it
will damage the filter bed in the cartridge and it will not work properly.
A) PREPARING THE SOFT DRINK
1. Measure 20mls of the soft drink using a measuring cylinder and put it into the small
100ml beaker.
2. Add 2-3 drops of the 0.7M TBAB (THE SMALL VIAL) into the beaker containing the
soft drink.
3. Mix using the glass rod.
4. Your soft drink is now ready to extract the colour.
B) PRIMING THE CARTRIDGE
1. Take the cartridge and fix the adaptor in
the top so it is tight.
The adaptor remains in this position
for the whole of the experiment.
2. Push the syringe into the top of the
adaptor so it, too, is snug.
3. Sit the assembly in the top of a boiling
tube.
4. Use a measuring cylinder to measure out
10 cm3 ethanol and pour it into the top of the syringe.
5. Now place the plunger into the syringe
6. Using the plunger, depress gently into the syringe expelling the used ethanol into the
boiling tube below.
7. Remove the syringe WITH THE PLUNGER STILL IN from the adaptor.
8. You can now remove the plunger from the syringe.
9. Put the syringe, minus the plunger, back into the adaptor.
10. Using a clean measuring cylinder measure out 10 cm3 of the 0.007M TBAB and pour
this into the top of the syringe.
11. Using the plunger, depress gently into the
syringe expelling the used 0.007M TBAB into
the boiling tube, as you did with the ethanol.
YOU HAVE PRIMED THE CARTRIDGE
AND IT IS READY TO EXTRACT YOUR
COLOUR FROM THE SOFT DRINK
Remove the syringe from the adaptor again and
then remove the plunger from the syringe.
Once again, reattach the syringe battle to the
adaptor.
C) EXTRACTING THE COLOUR FROM THE SOFT DRINK
1. Carefully pour 10 cm3 of your prepared coloured soft drink (20 cm3 of soft drink plus 2-3
drops of 0.007m TBAB) into the syringe barrel.
20 cm3 of soft
drink plus 2-3
drops of 0.007M
TBAB
2. Put in the plunger, push gently into the syringe, expelling the soft drink into the test tube
below.
3. Remove the syringe from the adaptor, then take out the plunger and re-attach the barrel
again. Put the second batch of drink in the top of the syringe and push that through as
before.
4. Remove the syringe and then take out the plunger before putting the syringe barrel back
in the adaptor.
REMEMBER TO REMOVE THE SYRINGE FROM THE ATTACHMENT BEFORE
REMOVING THE PLUNGER.
YOUR EXTRACTED COLOUR IS NOW ON THE CARTRIDGE.
D) CLEANING THE COLOUR ON THE CARTRIDGE
1. Ensure the syringe barrel is fixed to the
adaptor.
2. Sit it in the top of a boiling tube.
3. Use the measuring cylinder to measure out 10
cm3 of the 0.007M TBAB and pour it into the
top of the syringe.
4. Put in the plunger and push down gently on
the syringe expelling the used 0.007M TBAB
into the boiling tube below.
5. Remove the syringe from the adaptor and now remove the plunger from the syringe
YOUR COLOUR ON THE CARTRIDGE IS NOW CLEAN
E) TAKING THE EXTRACTED COLOUR OFF THE CARTRIDGE
1. You will need a CLEAN bijou.
2. Sit the cartridge (with the adaptor fixed in
the top of it) in the top of the bijou.
3. Measure 2 cm3 of ethanol using a pipette,
syringe or measuring cylinder and pour it
into the top of the syringe.
4. Put in the plunger and push down gently on
the syringe, expelling the extracted colour
into the bijou beneath.
THE EXTRACTED COLOUR IS IN THE
BIJOU AND IS READY TO BE
ANALYSED.
NB – the photographs show extraction into a
beaker – don’t worry, a bijou or a test tube
is just as good.
Analysis using paper chromatography
Chromatography is a technique used to separate
non-volatile mixtures.
It works by exploiting different interactions
between the dyes and the two components of the
chromatography setup.
There are two main components in
chromatography
A mixture of dyes is placed on some
chromatography paper and a solvent is allowed to
run up/along the paper carrying the different dyes
different distances.
The stationary phase –
this does not move and the liquids pass through it. (In ordinary
paper chromatography, the paper is the stationary phase.)
The mobile phase –
this is the fluid that moves through the stationary phase (like the
water through filter paper)
Notes
Pencil is always used to mark a chromatography paper because ink may run and interfere
with the chromatogram.
You may need to apply the colour for separation several times in order to get a strong enough
colour to separate well. If so, allow each one to dry before applying the next drop – otherwise
the drop will expand too much and blur the separation
Ensure the spot is dry before running the chromatogram. (Unless the solvent used for
extraction is the same as the mobile phase solvent) Otherwise the composition of the solvent
will be altered as it passes through the drop possibly compromising its effectiveness.
Make sure the spot is above the level of the solvent – otherwise the solvent will simply wash
it off into the solution.
It is important to make sure the solvent is level and not at an angle so you do not get an
angled solvent front.
Do not allow the sides of the paper to touch the sides of the container as that can make the
solvent front run crookedly.
Equipment and materials
Materials required by each student/group:
1 sheet of chromatography paper
500 – 600 cm3 beaker & ‘lid’
a rubber stopper for bottle with a slit about 3
mm deep along its narrow end
Capillary tubes
pencil
ruler
Chromatography solvent - sodium chloride
solution 0.1M
Samples of your extracted colours and
reference ones
Instructions
A Preparing the chromatography paper
1. Take the sheet of chromatography paper. Cut
the paper so that if fits into the beaker without
touching the sides (see diagram below)
2. Using a ruler, draw a light pencil line across the paper about 2.0cm from the bottom of
the sheet (see diagram below)
3. Draw dots or crosses along this line 2cm apart (see diagram below)
4. Mark above each dot the numbers 1-8, which correspond to the colour standard numbers
from the kit.
5. Leave the middle dot and mark as “sample”.
1
2
3
Dots or crosses
2cm apart
4
sample
5
6
7
Pencil line 2cm up
8
B spotting the chromatography paper
1. Using a capillary tube, apply a small spot of the unknown colour mixture (the extracted
colour in the beaker) on the pencil line at the centre dot on the paper. (The colour spot
should not exceed about 0.4 cm in diameter.)
2. If the spot seems to be too small or too light in colour, you can make it darker by
applying a second spot of colour (or more) directly on top of the dry first one.
(It is necessary to allow the spot to dry between applications of colour in order to keep it
small in size.)
3. Spot the paper with each of the available standard colours, allowing about 2.0 cm
minimum distances between each different spot. Ensure the colour standards you apply
match the number you have written in pencil at the top of the paper above each spot.
Prepared
chromatography
paper with spotting
The standards
R1
R2
R3
R4
R5
R6
E110 Sunset Yellow
E124 Ponceau 4R / E102 Tartrazine
E102 Tartrazine (+ 1% E129 Allura Red)
E129 Allura Red (+1% E102 Tartrazine )
Natural Red (Carmine or betelain)
Natural yellow (β-carotene or riboflavin)
C Preparing the solvents
1. Add 1% Sodium Chloride solution to a
clean 600 ml beaker, to a depth of about
0.5 cm (this will require approximately
25 ml of solution).
2. Bend the spotted chromatography paper
into a cylindrical shape, butt the ends
together (do not overlap the ends) and
staple them as shown.
D Running the chromatogram
1. Place the chromatography
paper into the beaker
making sure that the spots of
dye are NOT below the 1%
Sodium Chloride solution
level and that the paper is
NOT touching the sides of
the beaker.
Important – make sure
the spots you have made
are ABOVE the level of
the solution
2. Cover the beaker a watch glass. (or some clingfilm) (See diagram below)
Watchglass
covering beaker
3. Allow the 1% solution of Sodium Chloride to move up the paper to within 1 cm of the
top (this will take anywhere from 15 to 35 minutes).
E Collecting the results
1. Remove the paper from the beaker, open it flat, and, using a pencil, mark the solvent
front (the furthest level reached by the solvent).
solvent
front
Distance 1
outline
of spot
Distance 2
origin
2. Lay the chromatography paper on a paper towel to dry.
3. Outline each spot on the chromatography paper in pencil. Measure and record the
average distance from the spot or cross on the pencil line to the solvent front (shown
above as Distance 1).
Record your results.
Measure and record the distance each spot moved from the origin in the table below (Shown
above as distance 2).
This is used to work out the Rf values for each spot.
Remember you may have more than one spot in the unknown mixture and a
measurement in cm should be obtained for each.
Results
Spot
Average distance from
solvent front (cm)
Average distance from origin
(cm)
Ref 1
Ref 2
Ref 3
Ref 4
Sample 1
Sample 2
Sample 3
Sample 4
Ref 5
Ref 6
Ref 7
Ref 8
INTERPRETATION OF RESULTS
By matching both the visual colour and the Rf value of each of the spots, you can determine
the identity of the colours you extracted from the soft drink.
Save the chromatogram to hand in with your laboratory report.
CLEAN UP
Clean up all the glassware with soapy water.
Calculations
The Rf value for a spot is calculated using the formula:
The Rf value = Distance spot moved in cm / distance from origin to solvent front in cm
e.g. If your table shows
Ref 1
25
Then you Rf value will be 10/25 which will be 0.4
The Rf value will always be a decimal fraction.
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