Activity 1 – Practice
1. Read “Basic Method for Paper Chromatography” & “Spotting Methods”
2. Practice spotting food colours onto on a filter paper until you are able to
produce a small, dark spot. Try each of the following & see which you find
works best:




regular toothpick
catering (pointy) toothpick
capillary tube
pasteur pipette
3. Have a look at a piece of TLC sheet. Scrape a little of the silica gel off
the backing sheet with your thumbnail.
4. Try spotting food colours on a TLC sheet
Activity 2 – Comparison of 1M NaCl & Water in Separating Food Colours
1.
2.
3.
4.
Prepare a piece of chromatography paper for use in a 250ml beaker.
Spot each of three colours & a mixture of all colours onto a piece of paper.
Run the chromatography in either 1M Sodium Chloride or water.
Compare your results with someone who used the opposite solvent.
Activity 3 – Chromatography Using Sticks of Chalk
Chalk is a self supporting column that can be used for column chromatography.
Method
Draw a line around the chalk 2 cm from the larger end either with a mixture of food
colours or with a coloured texta. Stand the wider end in a Petri dish containing <1 cm
water. The water acts as a developing agent. The water is drawn up the column and
the component colours of the mixture are separated to produce different coloured
bands.
Activity 4 – Counterfeit Smarties?
Chromatography can be used to test some doubtful Smarties and compare them to
genuine Smarties.
ACTIVITY
Wash a smartie with 2-3 drops of water and a small paint brush in a small
watchglass. Then spot, using a capillary tube, toothpick or Pasteur pipette several
times. Compare various colours of the genuine Smarties with the suspect ones on a
strip of chromatography paper, in a 250ml beaker.
Activity 5 – Comparing Black Inks in a Forensic Science Unit
Preparation for this practical
Obtain 2 identical black (water soluble) textas.
Wearing gloves pull one texta apart & squeeze the ink from the central foam core.
With a little water wash as much additional ink as possible from the foam core & add
this to the extracted ink. If the resulting solution is too pale, leave the lid off in a
drafty area to allow to some of the water to evaporate.
Test this ink against a number of different black textas.
ACTIVITY
Using a small chromatography tank, paper clips & splints, run a comparison
chromatography of the extracted ink & 4 other black textas.
Activity 6 – Rf Values
ACTIVITY
Using the provided chromatogram calculating the Rf values of the spots & compare
with the table of Rf values for some common dyes.
The relationship of the distance moved by a pigment to the distance moved by the
solvent front is specific for a given set of conditions. (eg paper vs TLC & solvent
used)
Rf = Resolution Factor
Distance traveled by the pigment
Rf = --------------------------------------------------------Distance traveled by the solvent
RF values (Approximate) Colours and UV Fluorescence
25% Methylated spirits, 75% water, run on Chromatography paper.
Indicators
1-Naphthol
2- Naphthol
Alizarin Red S
Aniline Blue
Bromo Phenol Blue
Bromo Thymol Blue
Congo Red
Eosin, yellowish
Fluorescein
Indigo Carmine
Janus Green
Light Green
Methyl Orange
Methyl Red
Methyl Violet
Methylene Blue
Phenol Red
RF Range
0.54 - 0.63
0.41 – 0.57
0.80 - 0.99
0.63 - 0.86
0.83 - 0.90
0.80 - 0.86
0.00 - 0.02
0.58 - 0.70
0.71 - 0.78
0.76 – 0.84
0.00 - 0.23
0.86 - 0.99
0.62 - 0.74
0.69 - 0.76
0.23 - 0.35
0.05 - 0.19
0.79 - 0.83
RF
0.59
0.50
0.97
0.77
0.88
0.83
0.01
0.67
0.76
0.82
0.02
0.95
0.70
0.74
0.33
0.14
0.81
Colour
UV
Colourless
Light Blue
Colourless
Blue
Pink
Blue
Dark Blue
Dark Purple
Orange
Red
Pink
Orange/Pink Orange/Yellow
Yellow
Green
Blue
Dark Green
Light Green
Orange
Red/Pink
Purple
Blue
Yellow
Activity 7 – The Separation And Identification Of Photosynthetic
Pigments Using Thin-Layer Chromatography
INTRODUCTION
TLC usually gives a better separation of the components of a mixture than paper
chromatography. In this experiment we will use plastic silica gel sheets as the stationary
phase and a mixture of cyclohexane and ethyl acetate (5.5pts : 4.5pts) as the mobile phase,
to separate pigments in geranium leaves.
EQUIPMENT
pencil
ruler
TLC sheet 18 X 70 mm
several geranium leaves
scissors
mortar & pestle
small vial with lid
spatula
pinch acid washed sand
capillary tube
~ 5 ml acetone
75 X 25mm McCartney Bottle containing 2
ml Solvent (5.5 : 4.5 cyclohexane : ethyl
acetate)
METHOD
1.
Draw a thin pencil line across a TLC sheet about 1cm from one end.
2.
Remove the petioles (leaf stalks) from several geranium leaves and cut into 2-3 mm
pieces and place in a mortar with a little acid-washed sand ( ¼ rg) and grind to a
thick paste.
3.
Add about 1ml of acetone and grind, then add a further 2ml of acetone, grind and
pour the extract into the small vial and replace lid. (Work quickly as acetone
evaporates easily)
4.
Using a capillary tube containing the leaf extract, make a series of spots along this
line. Allow to dry and repeat twice. (cap the small vial while waiting for the line to dry)
5.
Place the TLC sheet in the McCartney bottle containing the solvent and replace lid.
6.
Observe the chromatogram as it develops.
7.
Remove the lid and take the TLC sheet out when the solvent front is about 0.5cm
from the top of the sheet.
8.
Mark the solvent front with a pencil.
9.
Make a labelled diagram of your chromatogram.
10.
Determine the Rf value of each pigment.
11.
Attempt to identify the pigments using the table below:-
Rf = Retardation Factor =
PIGMENT
Carotene
Phaeophytin
Chlorophyll a
Chlorophyll b
Xanthophyll a
Xanthophyll b
Xanthophyll c
Distance travelled by the pigment
--------------------------------------------------------Distance travelled by the solvent
COLOUR
Orange-yellow
Yellow-grey
Blue-green
Green-yellow
Yellow
Yellow
Yellow
Rf VALUE
0.96
0.81
0.75
0.70
0.51
0.37
0.17
Activity 7A – The Separation And Identification Of Photosynthetic
Pigments Using Thin-Layer Chromatography
INTRODUCTION
TLC usually gives a better separation of the components of a mixture than paper
chromatography. In this experiment we will use plastic silica gel sheets as the
stationary phase and a mixture of cyclohexane and ethyl acetate (5.5pts : 4.5pts) as
the mobile phase to separate pigments in spinach leaves. Consider the intermolecular forces between the pigments and the stationary and mobile phases.
EQUIPMENT
Spinach leaf in snap lock bag (frozen)
pencil
ruler
TLC sheet 20 X 60 mm
capillary tube
75 X 25mm McCartney Bottle
containing 2 ml Solvent (5.5 : 4.5
cyclohexane : ethyl acetate)
METHOD
1.
Collect a snap-lock bag of frozen spinach & while it is still frozen, crumble the
spinach until it is pulp. Using your fingers and with the bag still closed, try and
separate the liquid component from the solid.
2.
Draw a thin pencil line across a TLC sheet about 1cm from one end.
3.
Using a capillary tube and the liquid from the spinach, make a series of spots
along this line. Allow to dry and repeat twice.
4.
Place the TLC sheet in the McCartney bottle containing the solvent and
replace lid.
5.
Observe the chromatogram as it develops.
6.
Remove the lid and take the TLC sheet out when the solvent front is about
0.5cm from the top of the sheet.
7.
Mark the solvent front with a pencil.
8.
Make a labelled diagram of your chromatogram.
9.
Determine the Rf value of each pigment.
10.
Attempt to identify the pigments using the table below:
Rf = Retardation Factor =
PIGMENT
Carotene
Phaeophytin
Chlorophyll a
Chlorophyll b
Xanthophyll a
Xanthophyll b
Xanthophyll c
Distance travelled by the pigment
--------------------------------------------------------Distance travelled by the solvent
COLOUR
Orange-yellow
Yellow-grey
Blue-green
Green-yellow
Yellow
Yellow
Yellow
Rf VALUE
0.96
0.81
0.75
0.70
0.51
0.37
0.17
Activity 8 – Column Chromatography
For this practical normal (or calcined) Aluminium Oxide [Al2O3] will not work. Buy
Aluminium Oxide labelled “for Chromatography” [[Al2O3.xH2O].
As a Teacher Demo:
1. Using a long glass stirring rod or bamboo skewer insert a cotton wool plug
into the column & tap it down.
2. Mix a slurry of aluminium oxide and water & pour into the column.
3. Open the tap & allow some of the water to drain out but leave 2-3 mm of
water above the now compact aluminium oxide.
4. With the tap open add about 2 ml of a solution containing a mixture of
Potassium Permanganate and Potassium Dichromate.
5. When the solution has adsorbed into the column, add water initially in ~ 2ml
lots.
6. Continue to add water, so that the column never runs dry.
7. The Potassium Permanganate solution may be collected as it comes through
followed by the Potassium dichromate solution.
As a Student Experiment
This experiment can also be done as an individual experiment in a Pasteur pipette.
A small plug of cotton wool can be used instead of the glass wool & the pipette can
be held upright in a 10 ml measuring cylinder. The separate bands can be collected
on a welled tile.